JP3037554B2 - Immunogenic artificial polypeptide - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a polypeptide containing an antigenic site recognized by an antibody against a stem region in a hemagglutinin molecule of human influenza A virus and a gene encoding the polypeptide.

[0002]

BACKGROUND OF THE INVENTION There are three types of influenza viruses (A, B and C), which cause a worldwide pandemic of influenza and the death toll is due to human influenza A virus. Influenza A virus is further classified into many subtypes by the antigenicity of virus surface proteins haemagglutinin (hereinafter abbreviated as HA) and neuraminidase (hereinafter abbreviated as NA), and human influenza A virus is H1N1. Three types are currently known: subclass, H2N2 subclass, and H3N2 subclass. The HA of the influenza A virus has a bulbous region (
It consists of two different structures, head region and stem region. The bulb region contains the receptor binding site for the virus to bind to the target cells and
A is involved in the hemagglutinating activity of A, while the stem region contains the fusion peptide required for membrane fusion between the viral envelope and the endosomal membrane of the cell and is involved in the fusion activity [Wiley et al., Annual Review of Biochemistry (Ann. Rev. Biochem.), Vol. 56,
365-394 (1987)]. All anti-HA antibodies conventionally obtained as anti-HA antibodies recognizing the H1N1 subtype and H2N2 subtype recognize the spherical region of HA. However, this region is the most susceptible site for antigenic mutation, and therefore these antibodies do not commonly respond to the human influenza A virus subtype, and are associated with antigenic changes in the virus HA. The cognition is lost. On the other hand, Green et al. Synthesized a polypeptide from the amino acid sequence of the stem region of one type of HA of the H3N2 subtype and obtained antibodies against this polypeptide. The sum activity is weak (Tables 59-50)
No. 1714), and the polypeptide itself used as an antigen did not react with rabbit antiviral serum obtained by immunization with the H3N2 subtype, and had a problem in terms of antigenicity [Cell, No. 28, 477-487 (1982)]. The HA of influenza A virus is cleaved at one place by a protease,
Although the infectivity of the virus is activated, the larger polypeptide after the protease cleavage is HA1 and the smaller polypeptide is H1.
It is called A2. Of these, HA2 is said to have less antigenic variation due to the influenza A virus subtype. Grate H. (Glathe H.) et al. Disclose in East German Patent Application Publication No. 228737 that HA2 was extracted by subjecting virus particles to acid treatment followed by trypsin treatment or only treatment with a reducing agent. However, these operations destroy the three-dimensional structure of the HA molecule, resulting in an irreversible denatured state, and the resulting HA2 does not have the original three-dimensional structure. In addition, the above publication does not disclose that the obtained product was specifically confirmed as being effective as a vaccine.

[0003]

SUMMARY OF THE INVENTION Human Influenza A
Type virus periodically changes the type of HA and NA to cause a pandemic, and even if vaccinated before winter, the season of influenza, the effect of the vaccine is due to the spread of influenza caused by another type of virus. Is often not expected. However, if an antigen site in the HA molecule or NA molecule that is common to the virus subtypes and hardly causes antigen mutation can be obtained, this antigen site will be useful as a vaccine. An object of the present invention is to provide an antigen-site polypeptide which is recognized by an antibody having a recognizing property on the influenza A virus subtype and which can be used as a vaccine, and a gene encoding the polypeptide.

[0004]

SUMMARY OF THE INVENTION To summarize the present invention, the first invention of the present invention has the same antigenicity as the stem region in the HA molecule of human influenza A virus,
The present invention relates to an immunogenic artificial polypeptide in which a bulb region in a molecule has been deleted. Further, the second invention of the present invention relates to a gene encoding the immunogenic artificial polypeptide of the first invention of the present invention.

The present inventors have conducted intensive studies and have found that the H1N1 subtype and H2N
A polypeptide containing a common conserved antigenic site in each stem region in two subtypes of the HA molecule;
The present inventors have found that a polypeptide containing a commonly conserved antigenic site in a stem region in an HA molecule of the 3N2 subtype is extremely useful as a vaccine, The present inventors have completed the present invention by creating a gene encoding

The immunogenic polypeptide of the present invention has substantially the same antigenicity as the stem region in the HA molecule of the human influenza A virus and has a deletion of the globular region in the HA molecule. A polypeptide in which the globular region of the HA molecule has been engineered is typical, but the gene of the present invention obtained by specifically deleting the globular region from the gene encoding the HA molecule is referred to as the polypeptide of the present invention. It may be a polypeptide expressed by use. Furthermore, these polypeptides only need to have a three-dimensional structure that is recognized by an antibody that specifically recognizes a stem region in an HA molecule. May be added. In addition, during protein engineering production or genetic engineering production, it may be partially digested with protease. That is, in the present invention, having substantially the same antigenicity as the stem region in the HA molecule means that the stem region of the HA molecule has both the HA1 portion and the HA2 portion that can be effectively used as a vaccine. It means that you only need to. Therefore, the aforementioned Grate H. HA that has been denatured to destroy the original tertiary structure
A polypeptide consisting of only 2 is not included. In the present invention, examples of the most effective immunogenic artificial polypeptide for vaccines include the following. (1) at least a TGLRN polypeptide sequence represented by SEQ ID NO: 1 in the molecule;
Having a GITNKVNSVIEK polypeptide sequence represented by the formula: wherein the three-dimensional configuration of both sequences has substantially the same antigenicity as the stem region in the hemagglutinin molecule of the H1N1 subtype and H2N2 subtype. Artificial polypeptide. (2) at least a TGMRN polypeptide sequence represented by SEQ ID NO: 3 in the molecule;
Having a QINGKLNR (L / V) IEK polypeptide sequence represented by the formula: wherein the three-dimensional configuration of both sequences has substantially the same antigenicity as the stem region in the hemagglutinin molecule of the H3N2 subtype. Artificial polypeptide. (3) The artificial immunogenic polypeptide of the first invention of the present invention, which is isolated from a protease-treated product of a human influenza A virus hemagglutinin molecule.

H1N of human influenza A virus
An antibody that recognizes a common site in the HA molecular stem region of the H1N1 and H2N2 subtypes and neutralizes the virus,
A monoclonal antibody can be prepared as follows. For example, mammals such as mice, guinea pigs and rabbits are immunized with the following antigens. The following substances can be used as the antigen.

[0008] Virus particles selected from the H1N1 subtype or the H2N2 subtype can be used. As the H1N1 subtype, for example, A / Bangkok /
10/83, A / Yamagata / 120/86, A / Osaka
/ 930/88, A / Suita / 1/89 (above, Osaka University Institute of Microbial Diseases), A / PR / 8/34 [influenza (H1N1), ATCC VR-95], A
1 / FM / 1/47 [influenza A (H1N1),
ATCC VR-97], A / New Jersey / 8/76
[Influenza A (H1N1), ATCC VR-89
7], A / NWS / 33 [influenza A (H1N
1), ATCCVR-219], A / Weiss / 43 [influenza A (H1N1), ATCCVR-96],
A / WS / 33 [influenza A (H1N1), AT
CC VR-825]. Examples of the H2N2 subtype include A / Okuda / 57, A / Adachi / 2/57,
A / Kumamoto / 1/65, A / Kaizuka / 2/65, A
/ Izumi / 5/65 (preserved by the Institute for Microbial Diseases, Osaka University) and A2 / Japan / 305/57 [influenza A (H2N2), ATCC VR-100]. Alternatively, HA molecules obtained from these viruses, or HA polypeptides prepared using genetic recombination techniques, or a recognition site of the antibody of the present invention, that is, an antigen site of a stem region in the HA molecule is contained in the molecule. Immunization can be with a recombinant polypeptide or a synthetic polypeptide containing in its molecule an antigenic site in the stem region of the HA molecule.

Next, the spleen cells obtained from the immunized animal are fused with, for example, mouse myeloma cells, and cells that produce antibodies having the following properties (A) to (C) are selected from the obtained hybridomas. It can be prepared by culturing the cells. (A) It has a binding activity and a neutralizing activity to the viruses of the above-mentioned H1N1 subtype and H2N2 subtype. (B) H3N2 subtype virus, such as A / Fuku
oka / C29 / 85, A / Sichuan / 2/87, A / Ib
araki / 1/90, A / Suita / 1/90, A / Kitaky
ushu / 159/93 (above, Osaka University Research Institute for Microbial Diseases), A / PortChalmers / 1/73 [influenza A (H3N2), ATCC VR-810], A2 /
Aichi / 2/68 [Influenza A, ATCC VR
-547], and B-type viruses, such as B / Nagasaki
/ 1/87 (preserved by Osaka University Research Institute for Microbial Diseases), B / Al
len / 45 [influenza B, ATCC VR-10
No. 2] has neither binding property nor neutralizing activity. (C) H of H1N1 subtype and H2N2 subtype
Although it recognizes the A molecule, it does not inhibit the hemagglutination activity involving the bulb region in the HA molecule, and inhibits the fusogenic activity involving the stem region in the HA molecule.

The preparation of the hybridoma is carried out based on Nature, Vol. 256, pp. 495-497 (1975). Balb for immunization mice
/ C mice, F1 between Balb / c mice and other mice
A mouse or the like is used. Immunization was performed on one mouse
For example, using virus particles (100 to 1000 HA units) as an antigen, for example, three times in 2 to 5 months. The breeding of the mouse and the collection of the spleen cells are carried out according to a conventional method.

[0011] Myeloma cells include SP2 / 0-Ag
14 (ATCC CRL1581), p3 × 63Ag8
U. 1 (ATCC CRL1597), p3 × 63Ag
8 (ATCC TIB9), p3 × 63-Ag8.65
3 (ATCC CRL1580) and the like are preferably used. Spleen cells and myeloma cells were mixed at a ratio of 1: 1 to 10: 1, and fusion was performed using NaCl (about 0.85%), dimethyl sulfoxide [10 to 20% (v / v)] and a molecular weight of 1
This is performed by incubating the mixture of both cells at 35-37 ° C. for 1-5 minutes in a phosphate buffer (pH 7.2-7.4) containing 000-6000 polyethylene glycol. For the selection of the fused cells, HAT medium is used to select the cells that grow. Cloning of the fused cells is performed at least three times by the limiting dilution method.

If the hybridoma is cultured in the same manner as a normal animal cell, the antibody of the present invention can be obtained in the medium. In addition, the antibody of the present invention can be accumulated in ascites by transplanting the hybridoma into the abdominal cavity of a pristane-treated nude mouse or Balb / c mouse and growing it. That is, pristane 0.5~1mg inoculated into the mice intraperitoneally, the abdominal cavity transplanting 5 × 10 ⁶ ~1 × 10 ⁷ hybridomas subsequent 2-3 weeks. Usually ascites accumulates after 7 to 10 days and is collected. Monoclonal antibodies in culture and ascites fluid are purified by conventional means.

The obtained monoclonal antibody recognizes the stem region of the HA molecule of the H1N1 subtype and H2N2 subtype and neutralizes the virus by inhibiting the membrane fusion activity of the virus. It has properties.

(A) MDCK infected with H1N1 subtype and H2N2 subtype, respectively,
Recognizes cells (ATCC CCL34) but not MDCK cells infected with the H3N2 subtype. The staining test was performed using four types of antibodies (monoclonal antibody of the present invention, rabbit anti-mouse immunoglobulin G serum, goat anti-rabbit immunoglobulin G serum, and peroxidase-rabbit anti-peroxidase complex), and the Journal of Clinical Microbiology (J. Clin. Microbiol),
Vol. 28, pp. 1308-1313 (1990).

(B) H1N1 and H2N2 subtype HA molecules are recognized by immunoprecipitation
It does not recognize HA molecules of the N2 subtype.

(C) In the hemagglutination test, the hemagglutination activity of each of the H1N1, H2N2, and H3N2 subtypes is not inhibited.

(D) Recognizing a common conserved region specific to the stem region in the H1N1 subtype and H2N2 subtype HA molecule specified by the analysis of the gene encoding the HA molecule, and recognizing the H3N2 subtype HA molecule The common storage area specific to the trunk area is not recognized.

The analysis of the gene encoding the HA molecule is performed as follows. MDCK cells are infected with virus particles, the infected cells are collected the next day, and viral RNA is extracted from the cells using guanidine isothiocyanate. Next, H1
(-) Chain RN of each of N1, H2N2, and H3N2 subtypes
An oligonucleotide primer containing a sequence complementary to the 3 'end of A, for example, primer 5 represented by SEQ ID NO: 5 in the sequence listing is prepared, and cDNA is prepared using the primer.
Are synthesized. To amplify the cDNA, H1N1,
An oligonucleotide primer containing a sequence complementary to the 3 'end of the (+) strand RNA of each subtype of H2N2 and H3N2, for example, primer 6 represented by SEQ ID NO: 6 in the sequence listing was prepared, and the primer and primer 5 were prepared. PCR (Polymerase chain reaction)
The method allows efficient amplification. Amplified DNA
Approximately 1.7 kbp of the HA gene was separated by agarose gel electrophoresis.
After carrying out R, the amplified DNA is centrifuged using 20% (w / v) polyethylene glycol 6000 / 2.5 M NaCl to obtain a purified precipitate fraction. Next, a sequence primer selected from the HA gene sequence of each subclass of the virus was prepared, and the primer was labeled with [γ- ³² P] ATP. Then, the labeled primer and the purified fraction were annealed, The dideoxy method using a thermal cycler [BioTechniques, Vol. 9,
66-72 (1990)].

For example, primers 7 to 14 represented by SEQ ID NOs: 7 to 14 in the sequence listing are sequence primers for the H1N1 subtype, respectively.
The primers 15 to 23 represented by 5 to 23 are H
A sequence primer for the 2N2 subtype,
The primers 24-26 represented by SEQ ID NOs: 24-26 in the sequence listing are sequence primers for the H3N2 subtype. By using primers 9 and 13 as PCR primers and using primers 11 and 12 as sequence primers,
A part of the gene encoding the stem region of the HA molecule of the N1 subtype is efficiently amplified and analyzed. In addition, PCR
Using primers 17 and 21 as primers for use and primers 19 and 20 as sequencing primers, a part of the gene encoding the stem region of the HA molecule of the H2N2 subtype is efficiently amplified,
Is parsed. Further, by using primers 24 and 26 as PCR primers and using primers 25 and 26 as sequence primers, H3N
A part of the gene encoding the stem region of the two subtypes of the HA molecule is efficiently amplified and analyzed.

The common conserved region in the HA molecule between the virus subtypes is represented by SEQ ID NO: 1 in the sequence listing of the stem region in the HA molecule of the H1N1 subtype and H2N2 subtype discovered by the present inventors. And the GITNKVNSVIEK polypeptide sequence represented by SEQ ID NO: 2 in the Sequence Listing. FIG. 1 shows a schematic diagram of the tertiary structure of the HA molecule [Wiley et al., Nature, No. 289.
Pp. 373-378 (1981)], and H1N1.
The position of the common conserved region in the HA molecule between the subtype and the H2N2 subtype is shown. As shown in FIG. 1, both polypeptide sequences indicated by the A region and the B region are located close to each other at the center of the stem region of the HA molecule.
The monoclonal antibody C179 produced by oma C179 (FERM BP-4517) is composed of the TGLRN polypeptide sequence represented by SEQ ID NO: 1 in the sequence listing of the stem region in the HA molecule and GITNKVNSVIEK represented by SEQ ID NO: 2 in the sequence listing. Recognize the polypeptide sequence.

(E) In the neutralization activity test, the plaque-forming ability or the focus-forming ability of the H1N1 subtype and the H2N2 subtype is inhibited, and the plaque-forming ability or the focus-forming ability of the H3N2 subtype is not inhibited. The neutralization activity test is performed by the plaque reduction neutralization test or the influenza virus rapid focus reduction neutralization test described in the aforementioned Journal of Clinical Microbiology. That is, the antibody and the virus are mixed,
After incubating for a certain period of time, MDCK cells are infected, and the neutralizing ability is determined from the decrease in the appearance of plaques or foci.

(F) In the fusion activity test, the membrane fusion activity of the H1N1 subtype and H2N2 subtype is inhibited, but the membrane fusion activity of the H3N2 subtype is not inhibited. Fusion activity test is Nature, Vol. 300, 658-65
The method is performed according to the method described on page 9 (1982). That is, CV
After infecting -1 cells (ATCC CCL70) with the virus, antibody treatment was performed, and the ability to inhibit fusion activity was determined by the presence or absence of polykaryon formation.

The monoclonal antibody C179 binds to the stem region in the HA molecule, inhibits the membrane fusion activity of the H1N1 and H2N2 subtypes, and strongly neutralizes the infectivity of the virus strain. Thus, it binds to the stem region in HA molecules of the H1N1 and H2N2 subtypes,
A polypeptide that inhibits the membrane fusion activity of the H1N1 subtype and H2N2 subtype and induces an antibody (hereinafter, abbreviated as C179 type antibody) that strongly neutralizes the infectivity of the virus is used as a vaccine for influenza. Can be. That is, when a polypeptide having substantially the same antigenicity as the stem region in the HA molecule of the H1N1 subtype and H2N2 subtype and inducing a C179 antibody is used as an immunogen, the H1N1 subtype and the H2N2 subtype Prevent and treat influenza epidemics. As the immunogenic polypeptide, an HA molecule prepared from the H1N1 subtype or H2N2 subtype or an HA polypeptide prepared by a genetic recombination technique can be used. A polypeptide containing a stem region in an HA molecule as a component (hereinafter, abbreviated as a stem region polypeptide), in which a globular region in an HA molecule is easily deleted, is an immunogen that specifically induces a C179 type antibody. It is particularly effective as an active polypeptide.

The stem region polypeptide may be an HA molecule or an H molecule.
The globular region of the A polypeptide can be obtained by enzymatically limited decomposition and removal. For example, HA purified from virus particles of H1N1 subtype or H2N2 subtype
The stem region polypeptide can be prepared by subjecting a molecule to limited degradation with a protease. The stem region polypeptide may be prepared and used by treating the virus particles, the split vaccine obtained by inactivating the virus particles, and the extract obtained by treating the virus particles with a surfactant, respectively, with a protease.

As the protease to be used, a proteinase which can degrade the globular region without losing the antigenicity of the stem region in the HA molecule is desirable. As a proteinase that can be used in the present invention, proteinase K (EC 3.4.2.11.), Which is an alkaline proteinase produced by Tritirachium album, is used.
14), (made by Boehringer). The stem region polypeptide of the present invention can be prepared using a proteinase showing the same results as proteinase K. In addition, you may combine a proteinase and a peptidase, and may perform a peptidase process after a proteinase process. In addition, since HA molecules form a strong trimer in a solution, they are hardly subjected to protease digestion.
HA molecules in the presence of denaturing agents such as guanidine hydrochloride and urea
By treating the molecule with protease, H
Protease treatment of the A molecule can be performed. The concentration of the denaturant may be any concentration at which irreversible denaturation of the target stem region polypeptide does not occur and at which the protease also acts. When urea is used as a denaturing agent, 0.1 to 8
Protease digestion may be performed in the range of M, preferably 1-3 M of urea. The protease treatment can also be performed using a resin in which a protease is immobilized on a resin such as Sepharose, and after the reaction is completed, the protease-immobilized resin can be easily removed by centrifugation. The denaturant and low molecular substances in the reaction solution can be removed by dialysis, and a HA-treated protease can be prepared. The molecular weight of the protease-treated HA molecule can be measured by performing gel electrophoresis.

Further, by measuring the binding property between the protease-treated product and the C179-type antibody and the hemagglutinating activity of the protease-treated product, the target stem region polypeptide can be confirmed. The stem region polypeptide obtained by the protease treatment has substantially the same antigenicity (binding property to the C179 antibody) as the stem region in the HA molecule, and lacks the biological activity (hemagglutination activity) of the globular region. The lost polypeptide, which is composed of a polypeptide portion derived from the stem region of HA1 in the HA molecule and a polypeptide portion derived from HA2. In this regard, Grate H. composed of the HA2-derived polypeptide described above. Essentially different from these vaccines.

Further, a polypeptide having substantially the same antigenicity as the stem region in a natural HA molecule and having a deletion of the globular region in the HA molecule is prepared by a genetic recombination technique or a chemical synthesis method. , May be used. For example, from the RNA of H1N1 or H2N2 subtype virus, HA
A target polypeptide is obtained by preparing a gene, cutting and removing a site encoding the globular region in the gene with a restriction enzyme, integrating the vector into a vector, and expressing the vector in an animal cell, for example, a CV-1 cell. be able to. Further, after the HA gene has been incorporated into the vector, the site encoding the globular region in the HA gene can be selectively deleted by PCR, and animal cells, using a vector containing the gene, For example, the target polypeptide can be obtained by expressing the stem region polypeptide in CV-1 cells. The antigenicity of these stem region polypeptides can be confirmed by the binding properties of the C179-type antibody. The stem region polypeptide includes H1N in the molecule.
Any type may be used as long as it has a common conserved region in the stem region in HA molecules of the 1 subtype and the H2N2 subtype, and can induce a C179 type antibody. Examples of the stem region polypeptide having a common conserved region include a TGLRN polypeptide sequence represented by SEQ ID NO: 1 in the molecule and a GITNKVNSVIEK polypeptide sequence represented by SEQ ID NO: 2 in the molecule. Polypeptides whose sequence configuration is that of a native HA molecule that has substantially the same antigenicity as these sequences may be prepared and used. In addition, as the stem region polypeptide, amino acid deletion, substitution, insertion, transposition, addition or rearrangement may be used as long as the immunogenicity is not affected.For example, the C-terminal site of the stem region polypeptide and A polypeptide in which a part of the N-terminal polypeptide is deleted may be used, or a signal peptide of the HA molecule or a part of the globular region may be added.

H3N of human influenza A virus
An antibody that specifically recognizes a common site in the two subtypes of the HA molecule stem region can be prepared as a monoclonal antibody as follows. For example, mammals such as mice, guinea pigs and rabbits are immunized with the following antigens. The following substances can be used as the antigen. H3N2
Virus particles selected from subtypes can be used. Alternatively, HA obtained from these viruses
H prepared using molecular or genetic recombination techniques
A polypeptide or a recombinant polypeptide containing in its molecule a recognition site of the antibody of the present invention, that is, a stem region in an HA molecule, or containing a stem region in a HA molecule in its molecule It can be immunized with a synthetic polypeptide.

Next, the spleen cells obtained from the immunized animal were
For example, it can be prepared by fusing with a mouse myeloma cell, selecting a cell producing an antibody having the following properties (D) to (F) from the obtained hybridoma, and culturing the cell. (D) It has a binding property to the H3N2 subtype virus. (E) No binding to H1N1 and H2N2 subtype viruses. (F) H3N2 subtype HA molecules are recognized,
Hemagglutination activity involving the bulbous region in the A molecule is not inhibited.

The obtained monoclonal antibody recognizes the stem region of the HA molecule of the H3N2 subtype, and has the following properties in detail.

(G) In the staining test, MDCK cells infected with the H3N2 subtype (ATCC CCL34) are recognized, and MDCK cells infected with the H1N1 subtype and H2N2 subtype are not recognized.

(H) H3N2 subtype HA molecules are recognized by immunoprecipitation, and H1N1 subtype and H2N2
It does not recognize HA molecules of the N2 subtype.

(I) In the hemagglutination test, the hemagglutination activity of each of the H1N1, H2N2, and H3N2 subtypes is not inhibited.

(J) Recognize a common conserved region specific to the stem region in the HA molecule of the H3N2 subtype, which is specified by analysis of the gene encoding the HA molecule, and The common storage area specific to the trunk area is not recognized.

The common conserved region in the H3N2 subtype HA molecule includes the TGMRN polypeptide sequence represented by SEQ ID NO: 3 in the sequence listing of the stem region in the H3N2 subtype HA molecule discovered by the present inventors. And QINGKLNR (L / V) IEK polypeptide sequence represented by SEQ ID NO: 4 in the sequence listing. FIG. 2 shows a schematic diagram of the tertiary structure in the HA molecule [Willy et al., Supra], and the location of the common conserved region in the HA molecule of the H3N2 subtype. As shown in FIG. 2, both polypeptide sequences represented by A 'region and B' region are located close to each other at the center of the stem region in the HA molecule, and are hybridoma AI3C (FERM BP) which is an example of an antibody. -4516)
Is a TGMRN represented by SEQ ID NO: 3 in the sequence listing of the stem region in the HA molecule.
QING represented by the polypeptide sequence and SEQ ID NO: 4 in the sequence listing
KLNR (L / V) IEK polypeptide sequence.

A polypeptide that induces an antibody (hereinafter abbreviated as AI3C antibody) that specifically recognizes a common conserved region specific to the stem region in the HA molecule of the H3N2 subtype may be used as a vaccine for influenza. Can be. That is, the use of a polypeptide that has substantially the same antigenicity as the stem region in the HA molecule of the H3N2 subtype and induces an AI3C type antibody as an immunogen prevents and treats the epidemic of H3N2 subtype influenza. be able to. The immunogenic polypeptide includes H3N2
HA molecules prepared from subtypes and HA polypeptides prepared by genetic recombination techniques can be used.
A stem region polypeptide comprising the stem region of the HA molecule from which the globular region of the molecule has been deleted is particularly effective as an immunogenic polypeptide that specifically induces an AI3C type antibody.

The stem region polypeptide may be an HA molecule or an H molecule.
The globular region of the A polypeptide can be obtained by enzymatically limited decomposition and removal. For example, the stem region polypeptide can be prepared by subjecting an HA molecule purified from virus particles of the H3N2 subtype to limited degradation with a protease. The stem region polypeptide may be prepared and used by treating the virus particles, the split vaccine obtained by inactivating the virus particles, and the extract obtained by treating the virus particles with a surfactant, respectively, with a protease. As a protease to be used, a proteinase capable of degrading the globular region without losing the antigenicity of the stem region in the HA molecule is desirable. Examples of the proteinase that can be used in the present invention include the aforementioned proteinase K. The stem region polypeptide of the present invention can be prepared using a proteinase showing the same results as proteinase K. In addition, you may combine a proteinase and a peptidase, and may perform a peptidase process after a proteinase process. In addition, since HA molecules take a strong trimer in a solution, they are less susceptible to protease digestion,
Therefore, by subjecting the HA molecule to protease treatment in the presence of a denaturant such as guanidine hydrochloride or urea, the HA molecule can be efficiently treated with protease. The concentration of the denaturant may be any concentration at which irreversible denaturation of the target stem region polypeptide does not occur and at which the protease also acts. When urea is used as a denaturing agent, protease digestion may be performed in the presence of 0.1 to 8M, preferably 1 to 3M urea. The protease treatment can also be performed using a resin in which a protease is immobilized on a resin such as Sepharose, and after the reaction is completed, the protease-immobilized resin can be easily removed by centrifugation. The denaturant and low molecular substances in the reaction solution can be removed by dialysis, and a HA-treated protease can be prepared. The molecular weight of the protease-treated HA molecule can be measured by performing gel electrophoresis.

The target stem region polypeptide can be confirmed by measuring the binding between the protease-treated product and the C179-type antibody and the hemagglutinating activity of the protease-treated product. The stem region polypeptide obtained by the protease treatment has substantially the same antigenicity (binding to the AI3C type antibody) as the stem region in the HA molecule, and lacks the biological activity (hemagglutination activity) of the globular region. The lost polypeptide, which is composed of a polypeptide portion derived from the stem region of HA1 in the HA molecule and a polypeptide portion derived from HA2.

Further, a polypeptide having substantially the same antigenicity as the stem region in a natural HA molecule and having a deletion of the globular region in the HA molecule is prepared by a gene recombination technique or a chemical synthesis method. , May be used. For example, an HA gene is prepared from RNA of the H3N2 subtype virus, a site encoding the globular region in the gene is cleaved and removed with a restriction enzyme, and then incorporated into a vector, and used in animal cells such as CV-1 cells. By expression, the desired polypeptide can be obtained. Further, after the HA gene has been incorporated into the vector, the site encoding the globular region in the HA gene can be selectively deleted by PCR, and animal cells, using a vector containing the gene, For example, the target polypeptide can be obtained by expressing the stem region polypeptide in CV-1 cells.
The antigenicity of these stem region polypeptides can be confirmed by the binding of the AI3C type antibody.

As the stem region polypeptide, H in the molecule is used.
Any type may be used as long as it has a common conserved region in the stem region of the HA molecule of the 3N2 subtype and can induce an AI3C type antibody. Examples of the stem region polypeptide having a common conserved region include TG represented by SEQ ID NO: 3 in the molecule.
A MING polypeptide sequence and a QINGKLNR (L / V) IEK polypeptide sequence represented by SEQ ID NO: 4 in the Sequence Listing, wherein the configuration of the sequence is substantially identical to these sequences in the native HA molecule A polypeptide having a configuration having antigenicity may be prepared and used. In addition, as the stem region polypeptide, amino acid deletion, substitution, insertion, transposition, addition or rearrangement may be used as long as the immunogenicity is not affected.For example, the C-terminal site of the stem region polypeptide and And / or a part of the N-terminal polypeptide may be deleted, and a signal peptide of HA molecule,
A part to which a part of the spherical portion is added may be used.

When these polypeptides are used as vaccines, their antigenicity can be increased by selecting an appropriate carrier. As a carrier, for example, albumin, polyamino acid, etc. may be used. The vaccine of the present invention can be administered by a conventional active immunization system, that is, administered once or frequently in an amount which is effective for prevention, treatment and immunogenicity in a manner compatible with the dosage formulation. In addition, the vaccine may be formulated in a usual manner, and may contain an adjuvant for improving an immune response. The dose of the polypeptide to be administered depends on the nature of the vaccine to be used, the concentration in the preparation, the administration route and the like, but it is usually 1 μg to 100 mg, preferably 10 μg to 10 mg, for an adult.
Just give mg.

Hereinafter, the anti-influenza virus antibody used in the present invention will be described with reference examples. Reference example A1. Preparation of virus A / PR / 8/34, A / Ba as H1N1 subtype
ngkok / 10/83, A / Yamagata / 120/86, A
/ Osaka / 930/88, A / Suita / 1/89, Al
/ FM / 1/47 was used. A / Okuda / 57, A / Adachi / 2/57, A / Kumamo as H2N2 subtypes
to / 1/65, A / Kaizuka / 2/65, A / Izumi /
5/65 was used. A2 / Ai as H3N2 subtype
chi / 2/68, A / Fukuoka / C29 / 85, A / Si
chuan / 2/87, A / Ibaraki / 1/90, A / Suita
/ 1/90 and A / Kitakyushu / 159/93. B / Nagasaki / 1/87 was used as the B virus. Each virus was inoculated into the allantoic cavity of 11-day-old embryonated chicken eggs, and cultured at 34 ° C. for 4 days, and then each virus was collected.

2. Preparation of Monoclonal Antibody (1) The A / Okuda / 57 virus (320 HA units) prepared in Reference Example A-1 was suspended in Freund's complete adjuvant before use, and intraperitoneally injected in Balb / c mice at monthly intervals. One month after that, the mice were boosted by intraperitoneal injection using a PBS suspension of the same antigen (320 HA units). Three days later, the spleen was excised from the mouse to prepare splenocytes. P3 as mouse myeloma
X63Ag8 was cultured in a DME medium supplemented with 10% fetal bovine serum for 2 days after subculture, and washed with physiological saline before cell fusion to prepare. Next, the spleen cells and myeloma cells are mixed at a cell ratio of 1: 5, centrifuged to remove the supernatant, and the precipitated cell mass is sufficiently loosened.
Mixed solution [polyethylene glycol-4000 (2
g), MEM (2 ml), dimethyl sulfoxide], and kept at 37 ° C. for 5 minutes. Then, MEM was slowly added so that the total amount of the solution became 10 ml. Next, after centrifugation,
The supernatant was removed and the cells were loosened gently. To this was added 30 ml of normal medium [PRMI-1640 plus 10% fetal calf serum], and the cells were gently suspended using a female pipette.

The suspension was dispensed into a 96-well culture plate,
The cells were cultured at 37 ° C. for 24 hours in an incubator containing 5% CO ₂ . Next, a HAT medium was added, and the cells were cultured for 10 to 14 days. Subsequently, a portion of the culture supernatant was taken and hybridoma screening was performed.

(2) In order to obtain a monoclonal antibody that cross-reacts between influenza A virus subtypes, the above-mentioned culture supernatant, which has not been diluted, is used as a primary antibody, and three subtypes (H1N1, H2N2, H3N
A staining test of MDCK cells infected with each of 2) was performed. The staining test was carried out according to the method described in the above-mentioned Journal of Clinical Microbiology. That is, a 96-well microtiter plate (Falcon 30)
72: Becton Dickinson Co., Ltd.), each subtype strain of human influenza A virus (H1N1: A)
/ Yamagata / 120/86, H2N2: A / Okuda / 5
7. MDCK cells infected with H3N2: A / Fukuoka / C29 / 85) were washed with PBS (pH 7.4) and fixed with absolute ethanol at room temperature for 10 minutes. Next, these cells were treated with four kinds of antibodies [the culture supernatant containing the monoclonal antibody, a 1000-fold dilution of rabbit anti-mouse immunoglobulin G serum (manufactured by Organotechnica), and goat anti-rabbit immunoglobulin G serum (organotechnica). 500 times diluted solution of peroxidase-rabbit anti-peroxidase complex (manufactured by Organotechnica).
[00-fold dilution]], and the treated cells were washed with PBS for 40 minutes. Finally, the peroxidase reaction was carried out according to the method of Graham, Karnovsky (Garham, Karnovsky) using a PBS solution of 0.01% H ₂ O ₂ and 0.3 mg / ml of 3,3′-diaminobenzidine tetrahydrochloride [Journal Of histochemistry and cytochemistry (J. Histochem. Cytochem.), No. 14
Vol., Pp. 291-302 (1966)]. The stained cells were observed under a normal light microscope, and antibodies recognizing MDCK cells infected with the H1N1 subtype and MDCK cells infected with the H2N2 subtype were selected. Next, cells in the wells in which the cells in which the production of the antibody has been confirmed are growing are removed, subjected to a limiting dilution method three times, and the desired cells are cloned.
bridoma C179 and the monoclonal antibody produced by the hybridoma were designated as monoclonal antibody C179.

Hybridoma C179 is a hybridoma.
It has been deposited as FERM BP-4517 at the National Institute of Advanced Industrial Science and Technology, under the designation C179.

(3) The hybridoma strain was administered intraperitoneally to 5 × 10 ⁶ mice / animal to pristane-treated Balb / c mice. After 10 to 21 days, ascites was collected from the mouse in which ascites cancer was induced, and solid components were removed by centrifugation at 3000 rpm for 5 minutes to prepare ascites fluid. About 5 mg of the monoclonal antibody C179 (hereinafter simply referred to as C179) was contained in 1 ml of the ascites fluid. C179
Was purified by Protein A-Sepharose 4B (Pharmacia) and was an IgG2a type antibody.

3. Properties of Monoclonal Antibody (1) A 100-fold dilution of the ascites fluid described in Reference Example A-2- (3) was serially diluted, and the staining test described in Reference Example A-2- (2) was performed to recognize C179 antigen. The nature was examined. H1N1
A / PR / 8/34 and A / Bangkok as subtypes
/ 10/83, A / Yamagata / 120/86, A / Osak
a / 930/88, A / Suita / 1/89, A1 / FM
/ 1/47, A / Okuda / as H2N2 subtype
57, A / Adachi / 2/57, A / Kumamoto / 1/6
5, A / Kaizuka / 2/65, A / Izumi / 5/65,
A / Aichi / 2/68, A /
Fukuoka / C29 / 85, A / Sichuan / 2/87, A
/ Ibaraki / 1/90, A / Suita / 1/90, A / Ki
takyushu / 159/93, and B / Nagasaki / 1/87 as influenza B virus. C179
Recognized all H1N1 and H2N2 subtypes, but did not recognize H3N2 subtype or B virus.

(2) Neutralizing activity of the antibody was measured by the above-described influenza virus rapid focus reduction neutralization test, using the method described in Arches of Virol.
86, pp. 129-135 (1985), Microbiology and Immunology (Microbiol. I).
mmunol.), Vol. 29, pp. 327-335 (198).
Performed according to 5). Reference Example A-2-
Using the ascites fluid of (3), add 3 volumes of a solution of receptor-degrading enzyme (RDE: manufactured by Takeda Pharmaceutical Co., Ltd.) to the antibody before use, react at 37 ° C. for 18 hours, and heat at 56 ° C. for 45 minutes The RDE was inactivated by the treatment, and finally, it was prepared as a 16-fold diluted solution of ascites fluid, used as a test solution, and measured as follows.

That is, MDC was added to a 96-well microplate.
K cells were dispensed at 10 ⁴ cells / well and the next day, the antibody (16
An equal volume of a four-fold diluted solution (double dilution) and the respective virus solutions described in Reference Example A-3- (1) prepared in 30 focus forming units / hole were mixed and kept at 37 ° C. for 1 hour. Next, 25 μl of this mixture was dispensed into each well of the microtiter plate containing the MDCK cells, and then
The temperature was kept at 7 ° C. Next, the solution in each well was removed, each well was washed with PBS, and then MEM containing 0.5% tragacanth gum (Wako Pure Chemical Industries) and 5 μg / ml trypsin was added. After keeping the temperature at 37 ° C. for 20 to 24 hours, the added solution was removed, and each well was washed with PBS. Then, the cells were treated with absolute ethanol at room temperature for 10 minutes to fix the cells. Next, the cells were dried, and the cells were stained according to the staining test described in Reference Example A-2- (2). After staining, the cells were washed with tap water, dried, and the number of stained foci was counted under an optical microscope.

C179 inhibited the formation of foci of all H1N1 and H2N2 subtypes and showed strong virus neutralizing activity, but did not affect the formation of foci of H3N2 subtype and B virus. The same result was also obtained in the plaque reduction neutralization test.

(3) Hemagglutination-inhibiting activity (HI) of the antibody was performed as follows. RDE as in Reference Example A-3- (2)
A serial dilution was prepared using the treated antibody (32-fold dilution), and then each virus (1) described in Reference Example A-3- (1) was prepared.
6HA units) and reacted at room temperature for 30 minutes. Thereafter, chicken erythrocytes were added and mixed well, and the effect of the antibody on the hemagglutination activity of each virus was examined. C179
Had no effect on the hemagglutinating activity of all subtypes of virus.

(4) The measurement of the fusion inhibitory activity of the antibody was carried out as described in Nature, Vol. 300, pp. 658-659 (198).
The method of 2) was modified. That is, the monolayer culture of CV-1 cells was infected with each of the virus strains described in Reference Example A-3- (1). After 24 hours, the cells were washed twice with DMEM and then trypsinized (10 μl).
g / ml) in DMEM for 15 minutes at 37 ° C.
Next, after washing the cells twice with DMEM, Reference Example A-2-
Add a DMEM diluent of the ascites fluid of (3), and add
It was kept warm for 0 minutes. Thereafter, the cells were cultured in a fusion medium (PRMI without Na ₂ CO ₃ , 0.2% bovine serum albumin, 10 mM MES, 10 mM HEPE) adjusted to pH 5.0.
In S), the mixture was treated at 37 ° C. for 2 minutes, subsequently, washed twice with DMEM to remove the fusion medium, and then kept at 37 ° C. for 3 hours in DMEM containing 2% fetal bovine serum. Next, the cells were fixed with anhydrous methanol and stained with Giemsa, and then the presence or absence of polykaryon formation was determined with an optical microscope. C179
Inhibited polykaryon formation of all H1N1 and H2N2 subtypes, but not H3N2 subtype and B virus.

As described above, C179 is an H1N1 subtype,
The antibody specifically recognized the 2N2 subtype, inhibited virus membrane fusion, and exhibited neutralizing activity. Table 1 shows the results.

[0055]

[Table 1]

The numbers in the table are the dilution factor of the ascites fluid of Reference Example A-2- (3). Indicates the maximum dilution of the ascites fluid that can suppress the appearance of foci up to half of the number of foci in the control antibody-free group.
In addition, + means that polykaryon formation is completely inhibited by the antibody solution of the ascites fluid diluted 1000 times, and-means 10
This means that no inhibition was observed even when the doubling dilution of the ascites fluid was used. Further, no HI activity is observed even when the ascites fluid diluted 32 times is used.

4. Determination of Epitope (1) The recognition protein of C179 was determined to be an HA molecule by an immunoprecipitation reaction. That is, H2N2
After adsorbing and infecting the subtype A / Okuda / 57 on MDCK cells for 30 minutes, methionine in the medium was reduced to 10 μCi.
The cells were cultured for 24 hours in MEM substituted with “ ³⁵ S] methionine for labeling the infected cells.
PA buffer [50 mM Tris (pH 7.4), 150 mM N
aCl, 1 mM EDTA, 1% Nonidet P-40, 1
% Deoxycholic acid, 0.1% SDS]. Subsequently, after insoluble matter was removed by centrifugation, a supernatant was obtained. Next, the supernatant was mixed with C179, kept at 4 ° C. for 1 hour, added with protein A-Sepharose CL4B beads, kept at room temperature for 2 hours, and the immunoprecipitate was adsorbed to the beads. The beads are then collected and washed five times with RIPA buffer,
Boiling released the C179 binding protein. Next, the protein was subjected to SDS-12.5% polyacrylamide gel electrophoresis, the gel was fixed, immersed in 1 M sodium salicylate, dried, and subjected to autoradiography. The labeled protein bound to C179 was identified as an A / Okuda / 57 HA molecule from its electrophoresis pattern. Similar tests were performed for the H1N1 subtype, other H2
The test was performed using the N2 subtype and the H3N2 subtype, respectively. C179 has all H1N1 subtypes and H2
Although immunoprecipitated specifically with the HA molecule of the N2 subtype,
No binding was observed with the HA molecule of the H3N2 subtype.

(2) MDCK cells infected with the H1N1 subtype or H2N2 subtype were cultured in the presence of C179 to obtain an antigen mutant that was not sensitive to C179. That is, A / Su of H1N1 subtype as parent strain
ita / 1/89, A / Izumi / 5 of H2N2 subtype
/ 65, each of which was infected with the virus
K cells were cultured in the presence of C179, mutants capable of growing in the presence of C179 were isolated from plaques of MDCK cells, and A / Suita / 1/89 mutants were isolated from A / Su.
Mutants of ita / 1/89 (R) and A / Izumi / 5/65 were named A / Izumi / 5/65 (R), respectively. These two mutants showed no reactivity to C179 in the staining test and the neutralization test. In addition, this mutant strain was MD
The ability to form plaques on CK cells was also weak, it was not pathogenic to experimental animal mice, and was a weakly infected strain that could grow only on cultured cells.

(3) In order to identify the antigen recognition site of the antibody, a gene encoding an HA molecule (hereinafter abbreviated as HA gene) was analyzed. (A) Primer synthesis: Primer 5 to Primer 2
6 was synthesized using a DNA synthesizer, and after deprotection, ion exchange HPLC (TSK gel, DEAE-2SW column)
And desalted with Sep-pak C18 to obtain about 50 μg of each DNA.

(B) MDCK cells infected with A / Suita / 1/89 were collected, guanidine isothiocyanate was added, and the mixture was repeatedly injected and injected five times with a syringe to lyse the cells. Overlying the lysed cell extract on a cesium chloride solution,
Ultracentrifugation was performed. The precipitate at the bottom of the centrifuge tube was dissolved in a buffer, treated with phenol and chloroform, and then subjected to ethanol precipitation, and the recovered RNA was used as a sample of virus genomic RNA. Then, using primer 5, c
DNA was synthesized, followed by primer 5, primer 6
Was used to amplify the synthetic cDNA. Next, the amplified cDNA was separated by agarose gel electrophoresis, and a 1.7 kbp cDNA band corresponding to the HA gene was eluted. Using this cDNA with primers 5 and 6,
Further amplified by PCR method, amplified fragment was reduced to 20%
(W / v) polyethylene glycol 6000, 2.5M
A NaCl solution was added at 60% (v / v) and centrifuged to obtain a purified purified fraction.

Next, H labeled with [γ- ³² P] ATP
Using 1N1 subtype sequencing primers and primers 7-14, the nucleotide sequence of the purified gene was determined as follows by the dideoxy method using a thermal cycler described in Biotechnics. That is, 2
95 pmol primer and 1 pmol purified fragment
C. for 3 minutes and quenched for annealing. Next, tack polymerase was added, and 72 hours in a buffer containing deoxynucleotides and dideoxynucleotides.
The mixture was kept at a temperature of 10 ° C. for 10 minutes to perform a polymerase extension reaction.
Thereafter, the reaction was transferred to a thermal cycler in order to complete the extension reaction. Thermal cycler conditions are 90
A cycle of 1 minute at 55 ° C., 2 minutes at 72 ° C. for 3 minutes was performed 10 times. Next, the product after the cycle was heated at 95 ° C. for 3 minutes in the presence of formamide, and then quenched in ice, followed by electrophoresis on an 8% denaturing polyacrylamide gel. The gel after the electrophoresis is dried, exposed to an X-ray film, and the base sequence is read.
The nucleotide sequence of the entire A gene was determined.

(C) HA of A / Suita / 1/89 (R)
Analysis of the nucleotide sequence of the gene was performed according to Reference Example A-4- (3)-.
According to the method of (b), the nucleotide sequence of the entire HA gene was determined and compared with the HA gene of the parent strain. The mutant HA gene had three nucleotide substitutions. That is, H of the parent strain represented by SEQ ID NO: 27 in the sequence listing
G at base number 627 of the A gene corresponds to A, and base number 73
G at position 6 was mutated to A, and C at position 1018 was mutated to A. The HA molecule is cleaved at one place by a protease to activate the infectivity of the virus. The larger polypeptide after the protease cleavage is called HA1, and the smaller polypeptide is called HA2. Although these two polypeptides are connected by an SS bond, the mutations in this case are HA1, 189, 225 and 31.
Eighth was with an amino acid substitution. Amino acid residues 189 and 225 are in a highly variable region, and the 318th substitution, Thr → Lys (ACA → AAA at the nucleotide level), was responsible for the insensitivity of the mutant to C179. The amino acid numbers of the HA molecules in this specification are based on the H3 numbering method described in Virus, vol. 11, pages 257-266 (1961).

(D) A / Izumi / 5/65 and A / Izum
The analysis of the base sequence of each HA gene of i / 5/65 (R) was carried out according to the method of Reference Example A-4- (3)-(b), except that the primers and primers for sequencing the H2N2 subtype were used. 15 to 23 were performed. A /
The nucleotide sequence of the HA gene of Izumi / 5/65 is shown in SEQ ID NO: 28 in the sequence listing. The HA gene of the mutant had one nucleotide substitution. The T at base number 1197 of the HA gene of the parent strain represented by SEQ ID NO: 28 is mutated to A, and this mutation involves an amino acid substitution at the 52nd position of HA2, and the 52nd substitution, Val → Gl
u (GTA → GAA at the nucleotide level) was responsible for the inactivity of mutant C179.

(E) A / PR / 8 of H1N1 subtype
/ 34, A / Bangkok / 10/83, A / Yamagata / 1
20/86, A / Osaka / 930/88, H / N2 subtype A / Okuda / 57, A / Adachi / 2/57, A
/ Kumamoto / 1/65, A / Kaizuka / 2/65, H3
A2 / Aichi / 2/68 of N2 subtype, A / Fukuok
a / C29 / 85, A / Sichuan / 2/87, A / Ibar
H of HA molecule of aki / 1/90, A / Suita / 1/90
In order to identify the amino acid sequence near the 318th position of A1 and the amino acid sequence near the 52nd position of HA2, the base sequence of a part of each HA gene was analyzed.

Reference Example A for H1N1 subtype
RNA of each virus according to the method of -4- (3)-(b)
Genomic cDNA was synthesized, and PCR amplification was performed on the cDNA using primers 9 and 13. Using primers 11 and 12 with the obtained DNA fragment as a template,
The nucleotide sequence was determined by the dideoxy method using a thermal cycler.

For the H2N2 strain, refer to Reference Example A-4-
According to the method of (3)-(b), cDNA of the RNA genome of each virus was synthesized, and the cDNA was subjected to PCR amplification using primers 17 and 21. Obtained DN
The nucleotide sequence was similarly determined by the dideoxy method using primers 19 and 20 using the fragment A as a template.

Reference Example A-4-about H3N2 strain
CDNA for virus genomic RNA was synthesized according to the method of (3)-(b), and PCR amplification was performed on this cDNA using primers 24 and 26. Obtained DN
Using the A fragment as a template and primers 25 and 26, the nucleotide sequence was similarly determined by the dideoxy method.

The H1N1 subtype and the H2N2 subtype include 31 of the HA1 region represented by SEQ ID NO: 1 in the sequence listing.
The TGLRN polypeptide sequence of No. 8-322 (A region) and the GITNKVNSVIEK polypeptide sequence of No. 47-58 of the HA2 region represented by SEQ ID NO: 2 in the sequence listing (B region) are conserved, while H3N2 The subtype includes 318 of the HA1 region represented by SEQ ID NO: 3 in the sequence listing.
No.-322 TGMRN polypeptide sequence (A 'region)
And 47- of the HA2 region represented by SEQ ID NO: 4 in the sequence listing.
The QINGKLNR (L / V) IEK polypeptide sequence at position 58 (B 'region) is conserved. A region and A 'region are 1
Amino acids are different, and a difference of 5 to 6 amino acid residues is recognized between the B region and the B 'region. The difference between these two regions is the difference in antigen recognition of the antibody, and in serologic and fusion inhibition tests,
This is the reason that the antibody failed to react with the H3N2 subtype.

As shown in FIG. 1, the TGLRN polypeptide sequence represented by SEQ ID NO: 1 in the sequence listing for the A region and the GITNKVNSVIEK polypeptide sequence represented by SEQ ID NO: 2 in the sequence listing for the B region are represented by Located close to each other in the middle of the stem region, C179 recognizes both sequences, and this site is the epitope of C179. C179 is this H
It binds to the stem region of the A molecule, inhibits the membrane fusion function of the HA molecule, and neutralizes the virus.

H1N1 subtype: SEQ ID NO: 2 in Sequence Listing
The nucleotide numbers 1017 to 1031 of the HA gene of A / Suita / 1/89 shown in FIG.
191-1226 code the B area. SEQ ID NO: 29 in the sequence listing shows a part of the HA gene of A / PR / 8/34, and base numbers 76 to 90 encode the A region;
The base numbers 250 to 285 code for the B region. SEQ ID No. 30 in the sequence listing is A / Bangkok / 10/83
The base numbers 76 to 90 code for the A region, and the base numbers 250 to 285 code for the B region. SEQ ID No. 31 in the sequence listing is A / Yama
2 shows a part of the HA gene of gata / 120/86, and base numbers 76 to 90 encode the A region;
50 to 285 code the B region. SEQ ID No. 32 in the sequence listing shows a part of the HA gene of A / Osaka / 930/88, and base numbers 76 to 90 code for the A region, and base numbers 250 to 285 code for the B region. .

H2N2 subtype: SEQ ID NO: 2 in Sequence Listing
Nucleotide numbers 1007 to 1021 of the HA gene of A / Izumi / 5/65 shown in FIG.
Reference numerals 181 to 1216 code the B region. SEQ ID No. 33 in the sequence listing shows a part of the HA gene of A / Okuda / 57, and base numbers 94 to 108 thereof code for the A region;
The base numbers 268 to 303 code for the B region. SEQ ID No. 34 in the sequence listing is H for A / Adachi / 2/57
It shows a part of the A gene, and its base numbers 103 to 117 code for the A region, and its base numbers 277 to 312 code for the B region. SEQ ID NO: 35 in the sequence listing is A / Kuma
moto / 1/65 shows a part of the HA gene, and its base numbers 104 to 118 encode the A region, and its base number 2
78 to 313 code the B region. SEQ ID No. 36 in the sequence listing shows a part of the HA gene of A / Kaizuka / 2/65, and base numbers 88 to 102 code for the A region, and base numbers 262 to 297 code for the B region. .

H3N2 subtype: SEQ ID NO: 3 in Sequence Listing
7 is A2 / Aichi / 2/68, SEQ ID NO: 38 is A / Fuku
oka / C29 / 85, SEQ ID NO: 39 is A / Sichuan / 2
/ 87, SEQ ID NO: 40 shows a part of each HA gene of A / Ibaraki / 1/90, SEQ ID NO: 41 shows a part of each HA gene of A / Suita / 1/90, and base numbers 84 to 98 are A '.
And base numbers 258 to 293 each encode a B 'region.

[0073] 5. Preventive effect against influenza virus Influenza virus infection experiment in mice, C1
79 preventive effects were investigated. C to 10 Balb / c mice
179 PBS solution (1 mg / ml) was intraperitoneally administered at 1 ml per animal. A1 / FM / 1/47 of H1N1 subtype diluted 1000-fold after 1 day (4000 HA units)
Was inoculated intranasally in 25 μl portions. As a control, 12 mice were inoculated with virus only.

As shown in FIG. 3, 8 mice in the control group died (2 mice after 5 days, 5 mice after 6 days, 1 mouse after 8 days). Also, the other surviving mice were markedly weak. On the other hand, the mice to which C179 was administered were normal, and all were well even after 14 days. FIG. 3 is a diagram showing the survival rates of the C179-administered group and the non-administered group. The vertical axis represents the survival rate, and the horizontal axis represents the number of days after virus infection.

Reference Example B Preparation of virus As H5N3 subtype, A / whistling swan / shim
ane / 476/83, H / N6 subtype is A /
whistling swan / shimane / 37/80, A / turfted duck / shimane / 124R as H7N7 subtype
A / turky / Onta for / 80 and H8N4 subtypes
rio / 6118/68, A / chicken / Germany "N" / 49 as the H10N7 subtype, and inoculating each virus into the allantoic cavity of 11-day-old embryonated chicken eggs;
After culturing at 34 ° C. for 4 days, each virus was collected. Each of these virus strains is a strain preserved by the Research Institute for Microbial Diseases of Osaka University, and the HA molecule of A / chicken / Germany "N" / 49 has the amino acid sequences represented by SEQ ID NOs: 3 and 4 in the sequence listing, respectively. Have been.

2. Preparation of monoclonal antibody (1) A2 / Aichi / 2/68 prepared in Reference Example A-1
Of virus (320 HA units) was suspended in Freund's complete adjuvant before use, and immunized twice by intraperitoneal injection of Balb / c mice at monthly intervals. One month later, the same antigen (320 HA units) was used. The suspension was injected intraperitoneally using a PBS suspension and boosted. Three days later, the spleen was excised from the mouse to prepare splenocytes. Mouse myeloma
3x63Ag8 was cultured in DME medium supplemented with 10% fetal calf serum for 2 days after subculture, and washed with physiological saline before cell fusion to prepare. Next, the spleen cells and the myeloma cells are mixed at a cell ratio of 1: 5, centrifuged to remove the supernatant, and the precipitated cell mass is sufficiently loosened.
ml of a mixed solution [polyethylene glycol-4000 (2
g), MEM (2 ml), dimethyl sulfoxide], and kept at 37 ° C. for 5 minutes. Then, MEM was slowly added so that the total amount of the solution became 10 ml. Next, after centrifugation,
The supernatant was removed and the cells were loosened gently. To this was added 30 ml of normal medium [PRMI-1640 plus 10% fetal calf serum], and the cells were gently suspended using a female pipette.

The suspension was dispensed into a 96-well culture plate,
The cells were cultured at 37 ° C. for 24 hours in an incubator containing 5% CO ₂ . Next, a HAT medium was added, and the cells were cultured for 10 to 14 days. Subsequently, a portion of the culture supernatant was taken and hybridoma screening was performed.

(2) H3 of influenza A virus
In order to obtain a monoclonal antibody that reacts with the A 'region and the B' region conserved in the N2 type, the above culture supernatant, which has not been diluted, is used as a primary antibody, and three subtypes (H3N2, H10N7, H1N1) were stained for MDCK cells. The staining test was performed according to the method described in Reference Example A-2- (2). That is, a 96-well microtiter plate (Falcon 3)
072: Becton Dickinson) on the subtype strains of human influenza A virus (H3N2:
A2 / Aichi / 2/68, H10N7: A / chicken /
Germany "N" / 49, H1N1: A / PR / 8/3
MDCK cells infected with 4) were added to PBS (pH 7.4).
And then fixed with absolute ethanol at room temperature for 10 minutes. Next, these cells were treated with four types of antibodies [the culture supernatant containing the monoclonal antibody, a 1000-fold dilution of rabbit anti-mouse immunoglobulin G serum (manufactured by Organotechnica), and goat anti-rabbit immunoglobulin G serum (organotechnica). ) And a 1000-fold dilution of a peroxidase-rabbit anti-peroxidase complex (manufactured by Organotechnica)], and the treated cells were washed with PBS. Finally the peroxidase reaction, using 0.01% H ₂ O ₂ and 0.3 mg / ml of 3,3'-diaminobenzidine tetrahydrochloride in PBS, Graham, Karnofsky (Graham, Karnovsky)
Was performed in the manner described above. The stained cells were observed under a normal light microscope, and MDCK cells and H3N2 subtype-infected cells were examined.
Antibodies that respectively recognize MDCK cells infected with the 10N7 subtype were selected. Next, the cells in the wells where the production of the antibody has been confirmed are taken out, and the cells in the wells are taken out. The limiting dilution method is performed three times to clone the target cells, and the cloned hybridoma strain is hybridoma AI3C, which hybridoma produces. The monoclonal antibody was named monoclonal antibody AI3C. The product name of the monoclonal antibody AI3C is monoclonal antibody F49 (Takara Shuzo).

This Hybridoma AI3C is a hybridoma A
Denoted as I3C, it has been deposited with the National Institute of Advanced Industrial Science and Technology as a FERM BP-4516.

(3) The above hybridoma strain was intraperitoneally administered to pristane-treated Balb / c mice at 5 × 10 ⁶ mice / animal. After 10 to 21 days, ascites was collected from the mouse in which ascites cancer was induced, and solid components were removed by centrifugation at 3000 rpm for 5 minutes to prepare ascites fluid. About 1 mg of the monoclonal antibody AI3C (hereinafter simply referred to as AI3C) was contained in 1 ml of the ascites fluid. AI3C
Was purified with protein A-Sepharose 4B (Pharmacia).

3. Properties of Monoclonal Antibody (1) A 100-fold dilution of the ascites fluid described in Reference Example B-2- (3) was serially diluted and subjected to the staining test described in Reference Example A-2- (2) to recognize the antigen of AI3C. The nature was examined. H1N1
A / PR / 8/34 and A / Bangkok as subtypes
/ 10/83, A / Yamagata / 120/86, A / Osak
a / 930/88, A / Suita / 1/89, A1 / FM
/ 1/47, A / Okuda / as H2N2 subtype
57, A / Adachi / 2/57, A / Kumamoto / 1/6
5, A / Kaizuka / 2/65, A / Izumi / 5/65,
A / Aichi / 2/68, A /
Fukuoka / C29 / 85, A / Sichuan / 2/87, A
/ Ibaraki / 1/90, A / Suita / 1/90, A / Ki
takyushu / 159/93, and B / Nagasaki / 1/87 as influenza B virus, Reference Example B-
The virus described in 1 was also used. AI3C is all H3N
Two subtypes and A / chicken / German "N" / 49 were recognized, and the other subtype, B virus, was not recognized.

(2) The hemagglutination inhibitory activity (HI) of the antibody was determined as follows. RDE as in Reference Example A-3- (2)
A serial dilution was prepared using the treated antibody (32-fold dilution), and then mixed with each virus (16 HA units) described in Reference Example B-3- (1) and Reference Example B-1. At 30
Allowed to react for minutes. Thereafter, chicken erythrocytes were added and mixed well, and the effect of the antibody on the hemagglutination activity of each virus was examined. AI3C did not affect the hemagglutinating activity of all subtypes of virus.

Determination of Epitope (1) It was determined by immunoprecipitation that the AI3C recognition protein was an HA molecule. That is, H3N2
MDCK subtype A / Aichi / 2/68 for 30 minutes
After adsorbing and infecting cells, methionine in the medium was
The infected cells were labeled for 24 h in MEM substituted with μCi of “ ³⁵ S] methionine, and the cells were then collected and then RIPA buffer (50 mM Tris (pH 7.4), 150 mM).
NaCl, 1 mM EDTA, 1% Nonidet P-4
0.1% deoxycholic acid, 0.1% SDS]. Subsequently, after insoluble matter was removed by centrifugation, a supernatant was obtained. Next, the supernatant was mixed with AI3C, kept at 4 ° C. for 1 hour, added with protein A-Sepharose CL4B beads, kept at room temperature for 2 hours, and the immunoprecipitate was adsorbed to the beads. The beads were then collected, washed 5 times with RIPA buffer, and boiled to release AI3C binding protein. Next, the protein was subjected to SDS-12.5% polyacrylamide gel electrophoresis, the gel was fixed, immersed in 1 M sodium salicylate, dried, and subjected to autoradiography. From the electrophoresis pattern, the labeled protein to which AI3C binds was A2 / Aichi / 2/6.
Eight HA molecules were identified. Similar tests were performed using the H1N1 subtype, H2N2 subtype, other H3N2 subtypes, and the virus described in Reference Example B-1. AI3C is available for all H3N2 subtypes and A / ch
Although immunoprecipitated specifically with the HA molecule of icken / Germany "N" / 49, it did not show binding to HA molecules of the H1N1 and H2N2 subtypes.

[0084]

EXAMPLES The present invention will be described below with reference to examples, but the present invention is not limited to these examples.

Example 1 Preparation of Antigen Polypeptide (1) Synthesis of Primer: (-) of H2N2 Subtype
Oligonucleotide primers containing a sequence complementary to the 3 'end of the strand RNA (primer 27, primer 2
8) and an oligonucleotide primer containing a sequence complementary to the 3 'end of the (+) strand RNA (primer 2
9, primer 30) is synthesized using a DNA synthesizer,
After deprotection, ion exchange HPLC (TSK gel, DEA
E-2SW column) and purified by Sep-p
ak) Desalting with C18 to obtain about 50 μg of each DNA. The nucleotide sequences of the primers 27 to 30 are shown in SEQ ID NOs: 42 to 45 in the sequence listing.

(2) MD infected with A / Okuda / 57
CK cells were collected, guanidine isothiocyanate was added, injection was repeated five times with a syringe, and injection was repeated to lyse the cells. The cell extract after lysis was overlaid on a cesium chloride solution, and ultracentrifuged. The precipitate at the bottom of the centrifuge tube was dissolved in a buffer solution, treated with phenol and chloroform, and then precipitated with ethanol.
Sample. Next, using primer 5, cDNA
Then, the synthesized cDNA was amplified by PCR using primers 27 and 29. Next, the amplified cDNA was separated by agarose gel electrophoresis, and a 1.8 kbp cDNA band corresponding to the HA gene was eluted. This cDNA was further amplified by the PCR method using primers 28 and 30 to obtain a 1.8 kbp secondary amplification D
The NA fragment was converted to a 20% (w / v) polyethylene glycol 6000, 2.5 M NaCl solution at 60% (v / v).
v) In addition, the mixture was centrifuged to obtain a purified precipitate fraction.

(3) The nucleotide sequence of the HA gene of A / Okuda / 57 was analyzed according to the method of Reference Example A-4- (3)-(b, d). The nucleotide sequence and amino acid sequence of the HA gene of A / Okuda / 57 are shown in SEQ ID NO: 46 in the sequence listing. In SEQ ID NO: 46, base numbers 1 to 5 are sequences derived from primer 30, base numbers 6 to 48 are non-coding regions, base numbers 49 to 93 are signal peptide coding regions, and base numbers 94 to 231 are stem regions. A region encoding the N-terminal side, base numbers 232 to 873 are regions encoding the globular region, base numbers 874 to 1734 are regions encoding the C-terminal side of the stem region, base numbers 1735 to 1735
1775 is a non-coding region, base numbers 1776 to
1783 is the sequence derived from primer 30.

(4) Preparation of plasmid (a) The terminal of the 1.8 kbp secondary amplified DNA fragment prepared in Example 1- (3) was blunt-ended using T4 DNA polymerase. Next, the blunted fragment and the SmaI digest (2.7 kbp) of plasmid pHSG299 (manufactured by Takara Shuzo) were ligated with T4 DNA ligase to prepare a plasmid into which the HA gene had been incorporated. Next, Escherichia coli JM109 was transformed with the plasmid, and the plasmid was isolated from the resulting kanamycin-resistant transformant. The plasmid was named plasmid pH2-299.

The Escherichia coli JM109 strain transformed with the plasmid pH2-299 was Escherichiacoli JM109 / pH2-299.
It has been deposited with the National Institute of Advanced Industrial Science and Technology as a FERM P-13431.

(B) Plasmid pEF-BOS [Nucleic Acids Research
arch), Vol. 18, p. 5322 (1990)]
The 2.6 kbp B obtained by cutting the tII digest and the plasmid pMAM _neo-s (Clontech) with BamHI.
The amHI DNA fragment was ligated with T4 DNA ligase to prepare a plasmid of a shuttle vector for Escherichia coli and animal cells into which the 2.6 kbp BamHI DNA fragment was integrated. Next, Escherichia coli JM109 was transformed with the plasmid, a plasmid was isolated from the resulting ampicillin-resistant transformant, and the isolated plasmid was designated as plasmid pE.
It was named F-BOS / NeoA.

The plasmid pH2-299 was digested with NheI to prepare a 1.8 kbp DNA fragment having the sequence of nucleotide numbers from 4 to 1780 of the cDNA represented by SEQ ID NO: 46 in the sequence listing. Next, the DNA fragment and the XbaI digest (7.9 kbp) of the plasmid pEF-BOS / NeoA were ligated with T4 DNA ligase to prepare a shuttle vector in which the HA gene was incorporated. Next, Escherichia coli JM109 was
The strain was transformed, and a plasmid was isolated from the resulting ampicillin-resistant transformant. The plasmid was named plasmid pEBNaH2. Further, Escherichia coli JM109 strain transformed with the plasmid was used.
It was named / pEBNaH2.

(C) oligonucleotide primers represented by SEQ ID NOs: 47 and 48 in the sequence listing (primers 31,
Primer 32) was synthesized using a DNA synthesizer, and after deprotection, ion exchange HPLC (TSK gel, DEAE-
2SW column) and purified by Sep-pa
k) Desalting with C18 to obtain about 50 μg of each DNA. Primer 31 has a DNA sequence corresponding to the DNA sequence of base numbers 207 to 231 of the HA gene represented by SEQ ID NO: 46 in the sequence listing.
The primer is an antisense strand primer for RNA, and the primer 32 is a sense strand primer for mRNA corresponding to the DNA sequence of base numbers 874 to 899.

By using the primers 31 and 32, 3.8 kbp was removed from the plasmid pH 2-299 by removing only the region encoding the globular region of the HA gene.
Was attempted to amplify the DNA fragment by the PCR method. Es
cherichia coli JM109 / pH2-299 (FERM P-134
The plasmid pH2-299 was prepared from 31), and PCR was performed using 0.05 pmol of the plasmid, 50 pmol of each of the primers 31 and 32. PCR conditions are 90
A cycle of 1 minute at 55 ° C., 2 minutes at 72 ° C., and 3 minutes at 72 ° C. was performed 25 times to prepare an amplified DNA of 3.8 kbp.
Next, the 3.8 kbp DNA fragment was phosphorylated using T4 kinase, blunt-ended with T4 DNA polymerase, and ligated with T4 DNA ligase to prepare a circular plasmid. Next, E. coli J
M109 was transformed, and a plasmid was isolated from the resulting kanamycin-resistant transformant. The plasmid was designated plasmid p299H2S _nc.

[0094] By cutting the plasmid P299H2S _nc with NheI, than the HA gene, a DNA fragment of 1.1kbp which the region encoding the globular region (corresponding to nucleotide numbers 232 to 873 of SEQ ID NO: 46) has been removed prepared did. The nucleotide sequence of the fragment is represented by SEQ ID NO: 4 in the sequence listing.
9 shows the amino acid sequence encoded by the fragment as SEQ ID NO: 50 in the sequence listing. Next, the 1.1 kbp DNA fragment and the shuttle vector plasmid pEF-BOS /
NeoA XbaI digest (7.9 kbp) was ligated with T4 DNA ligase, and a plasmid into which a DNA encoding a region encoding the N-terminal side of the stem region of the HA gene and a region encoding the C-terminal side were directly integrated was used. Produced. Next, Escherichia coli JM109 was transformed with the plasmid, and the plasmid was isolated from the resulting ampicillin-resistant transformant. The plasmid was named pENH2dHO1.

The plasmid was transformed with pENH2dHO1 and the Escherichia coli JM109 strain was transformed into Escherichia coli JM109 / pENH2dHO1.
It has been deposited and designated as FERM BP-4190 by the Institute of Biotechnology and Industrial Technology, National Institute of Advanced Industrial Science and Technology.

(5) Expression of polypeptide: Escherichia
E. coli JM109 / pENH2dHO1 (FERM BP-4190) was used to prepare a plasmid pENH2dHO1 in which a gene encoding a polypeptide lacking the globular region of the HA molecule was prepared, and the HA molecule was encoded from Escherichia coli JM109 / pEBNaH2. Plasmid pEBNaH2 into which the gene was inserted was prepared. About 5 × 10 ⁶ CV-1 cells were treated with trypsin, washed once with 20 ml of 10% FCS-MEM (manufactured by Dulbecco), twice with PBS, and washed with 1 ml of PBS.
Suspended in water. 0.8 ml of the mixture was placed in a cuvette for GenePulser, a gene transfer device manufactured by Bio-Rad, 30 μg of plasmid pENH2dHO1 was added, and the mixture was stirred with a Pasteur pipette and allowed to stand at 0 ° C. for 10 minutes. Set the cuvette in the gene transfer device Gene Pulser,
After treatment under the conditions of V and 960 μFD, the cells were allowed to stand at 0 ° C. for 10 minutes to transform CV-1 cells. Next, the transformed cells are placed in 30 ml of 10% FCS-MEM, and
The cells were cultured in a petri dish with a diameter of 6 cm for 2 days. The cultured CV-1 cells were washed with PBS (pH 7.4) and fixed with absolute ethanol at room temperature for 10 minutes. Next, these cells were diluted with four kinds of antibodies [C179, rabbit anti-mouse immunoglobulin G serum (manufactured by Organotechnica) 1000-fold dilution, goat anti-rabbit immunoglobulin G serum (manufactured by Organotechnica) 500-fold dilution. , A 1000-fold diluted solution of peroxidase-rabbit peroxidase complex (manufactured by Organotechnica)], and the treated cells were washed with PBS. Finally, the peroxidase reaction is carried out according to the method of Graham, Karnovsky, using a PBS solution of 0.01% H ₂ O ₂ and 0.3 mg / ml of 3,3′-diaminobenzidine tetrahydrochloride. Was. C transformed with plasmid pENH2dHO1
V-1 cells stained brown and expressed a polypeptide in which the globular region of the HA molecule was deleted, but the higher order structure of the stem region was maintained normally.

Since the expressed polypeptide is likely to be an antigenic determinant and lacks a globular region in which an antigenic mutation is liable to occur, it specifically recognizes the H1N1 subtype and H2N2 subtype stem regions, and Can be used as a vaccine to induce an antibody that neutralizes the antibody.

Using plasmid pEBNaH2, CV-
One cell was transformed, the transformant was cultured, and a staining test of the cultured cells using C179 was performed. Plasmid pE
CV-1 cells transformed with BNaH2 are stained brown,
These cells also expressed an HA polypeptide having a normal higher-order structure in the stem region.

Example 2 Preparation of Antigen Polypeptide (1) Synthesis of Primer: H3N2 Subtype (-)
Oligonucleotide primer containing a sequence complementary to the 3 'end of the strand RNA (primer 33), and (+)
Oligonucleotide primers containing a sequence complementary to the 3 'end of the strand RNA (primer 34, primer 3
5) was synthesized using a DNA synthesizer, and after deprotection, purified by ion exchange HPLC (TSK gel, DEAE-2SW column), desalted with Sep-pak C18, and each DNA was purified. 50 μg were obtained. Primer 33 ~
SEQ ID NOS: 51 to 5 of the sequence listing
3 is shown.

(2) MDCK cells infected with A2 / Aichi / 2/68 were collected, guanidine isothiocyanate was added, the mixture was repeatedly injected and injected five times with a syringe, and the cells were lysed. Overlying the lysed cell extract on a cesium chloride solution,
Ultracentrifugation was performed. The precipitate at the bottom of the centrifuge tube was dissolved in a buffer, treated with phenol and chloroform, and then subjected to ethanol precipitation, and the recovered RNA was used as a sample of virus genomic RNA. Then, using primer 5, c
DNA was synthesized, and then the synthesized cDNA was amplified by PCR using primers 33 and 34. Next, the amplified cDNA was separated by agarose gel electrophoresis, and a 1.8 kbp cDNA band corresponding to the HA gene was eluted. Using this cDNA as primers 33 and 35
And a 1.8 kbp secondary amplified DNA fragment was converted to a 20% (w / v) polyethylene glycol 6000, 2.5 M NaCl solution at 60%.
(V / v) and centrifuged to obtain a purified purified fraction.

(3) Analysis of the nucleotide sequence of the HA gene of A2 / Aichi / 2/68 was carried out according to Reference Example A-4- (3)-(b,
It carried out according to the method of d). A2 / Aichi / 2/68 H
The nucleotide sequence and the amino acid sequence of the A gene are shown in SEQ ID NO: 54 in the sequence listing. In SEQ ID NO: 54, base numbers 1 to 8
Is a sequence derived from the primer 33, base numbers 9 to 36 are non-coding regions, base numbers 37 to 84 are regions encoding a signal peptide, base numbers 85 to 246 are regions encoding the N-terminal side of the stem region, base numbers 247-90
3 is a region encoding a globular region, base numbers 904 to 1
734 is a region encoding the C-terminal side of the stem region, base numbers 1735 to 1769 are non-coding regions, and base numbers 1770 to 1777 are sequences derived from primer 35.

(4) Preparation of plasmid (a) The end of the 1.8 kbp secondary amplified DNA fragment prepared in Example 2- (3) was blunt-ended using T4 DNA polymerase. Next, the blunted fragment and the HincII digest of plasmid pUC118 (Takara Shuzo) (1.8
kbp) was ligated with T4 DNA ligase and
A plasmid having the A gene integrated therein was prepared. Next, Escherichia coli JM109 was transformed with the plasmid, and the plasmid was isolated from the resulting ampicillin-resistant transformant. The plasmid was designated plasmid pU118H3 _xxn.

[0103] plasmid pU118H3 E. coli JM109 strain was transformed with _xxn Escherichia coli JM109 / pU118H3
_It is named and designated as _xxn, and is deposited with the National Institute of Advanced Industrial Science and Technology as a FERM P-13567.

(C) oligonucleotide primers represented by SEQ ID NOs: 55 and 56 in the sequence listing (primers 36 and
Primer 37) was synthesized using a DNA synthesizer, and after deprotection, ion exchange HPLC (TSK gel, DEAE-
2SW column) and purified by Sep-pa
k) Desalting with C18 to obtain about 50 μg of each DNA. The primer 36 has a DNA sequence corresponding to the DNA sequence of base numbers 227 to 246 of the HA gene represented by SEQ ID NO: 54 in the sequence listing.
The primer is an antisense strand primer for RNA, and the primer 37 is a sense strand primer for mRNA corresponding to the DNA sequence of base numbers 904 to 923.

[0105] By using the primer 36 and primer 37, tried to amplification by PCR of a DNA fragment of 4.3kbp excluding only the region encoding the globular region of the HA gene from the plasmid pU118H3 _xxn. Esch
erichia coli JM109 / pU118H3 _xxn (FERM P-13
567) Plasmid PU118H3 _xxn were prepared from, the plasmid 0.05Pmol, primer 36, a PCR using primers 37 each 50pmol was performed. PCR conditions are 90
A cycle of 1 minute at 55 ° C., 2 minutes at 72 ° C. for 3 minutes was performed 25 times to prepare a 4.3 kbp DNA amplified product.
Next, the 4.3 kbp amplified DNA fragment was phosphorylated using T4 kinase, blunt-ended with T4 DNA polymerase, and ligated with T4 DNA ligase to prepare a circular plasmid. Next, Escherichia coli JM109 was transformed with the plasmid, and the plasmid was isolated from the resulting ampicillin-resistant transformant. The plasmid was named plasmid p118H3dHO1 _xxn .

The plasmid p118H3dHO1 _xxn was _replaced with XbaI, N
By cutting with heI, a region encoding the globular region (base number 247 of SEQ ID NO: 54) was obtained from the HA gene.
(Corresponding to .about.903) was removed to prepare a 1.1 kbp DNA fragment. The nucleotide sequence of the fragment is shown in SEQ ID NO: 57 in the sequence listing, and the amino acid sequence encoded by the fragment is shown in SEQ ID NO: 58 in the sequence listing. Then this 1.1kb
p DNA fragment and the XbaI digest (7.9 kbp) of the shuttle vector plasmid pEF-BOS / NeoA were converted to T4
Ligation was performed with DNA ligase to prepare a plasmid in which a DNA in which a region encoding the N-terminal side of the stem region of the HA gene and a region encoding the C-terminal side were directly linked was integrated. Next, Escherichia coli JM109 was transformed with the plasmid, and the plasmid was isolated from the resulting ampicillin-resistant transformant. The plasmid was named pENH3dHO1.

The Escherichia coli JM109 strain transformed with the plasmid pENH3dHO1 was named and designated as Escherichia coli JM109 / pENH3dHO1, and deposited with the National Institute of Bioscience and Human Technology as FERM BP-4518.

(5) Expression of polypeptide: Escherichia
A plasmid pENH3dHO1 was prepared from E. coli JM109 / pENH3dHO1 (FERM BP-4518), into which a gene encoding a polypeptide lacking the globular region of the HA molecule was incorporated. About 5 × 10 ⁶ CV-1 cells were treated with trypsin, washed once with 20 ml of 10% FCS-MEM (Dalbeco), twice with PBS, and suspended in 1 ml of PBS.
0.8 ml of this was placed in a cuvette for Gene Pulser, a gene transfer device from Bio-Rad, and the plasmid pENH3d
30 μg of HO1 was added, stirred with a Pasteur pipette, and allowed to stand at 0 ° C. for 10 minutes. Set the cuvette in the gene transfer device Gene Pulser, 250 V, 960 μF
After treatment under the conditions of D, the mixture was allowed to stand at 0 ° C. for 10 minutes, and CV-
One cell was transformed. The transformed cells are then
Put in 0 ml of 10% FCS-MEM, 5 ml each, 6 cm in diameter
For 2 days.

The cultured CV-1 cells were transferred to PBS (pH 7.
After washing in 4), the cells were fixed with absolute ethanol at room temperature for 10 minutes. Next, these cells were diluted 1000 times with four kinds of antibodies (AI3C, rabbit anti-mouse immunoglobulin G serum (manufactured by Organotechnica), and 500-fold diluted solution with goat anti-rabbit immunoglobulin G serum (manufactured by Organotechnica). , A 1000-fold dilution of a peroxidase-rabbit peroxidase complex (manufactured by Organotechnica)], and the treated cells were washed with PBS. Finally, the peroxidase reaction was performed at 0.01% H. _Two
Using a PBS solution of O ₂ and 0.3 mg / ml of 3,3′-diaminobenzidine tetrahydrochloride, Graham
aham, Karnovsky). Plasmid pENH3d
CV-1 cells transformed with HO1 stained brown and expressed a polypeptide in which the globular region of the HA molecule was deleted, but the higher order structure of the stem region was retained normally. Because this expressed polypeptide is likely to be an antigenic determinant, and lacks the globular region where antigenic mutations are likely to occur,
It can be used as a vaccine that induces an antibody that specifically recognizes the stem region of the H3N2 subtype.

Example 3 Preparation of Antigen Polypeptide (1) Preparation of HA Molecule A / Yamagata / 32/89 virus particles (40 mg) prepared in Reference Example A-1 were added to 27 ml of 5 mM Tris-HCl.
(PH 8.0), 3 ml of 20% NP-40 was added, and the mixture was kept at 37 ° C. for 30 minutes. Next, after centrifugation, the supernatant was collected, and a 0.8 μm filter unit (Milex-
(PF: Millipore). Then
The filtrate was loaded on an ion exchange membrane (memSep DEAE: manufactured by Millipore) equilibrated with 5 mM Tris-HCl (pH 8.0), and washed with the same buffer. Further, HA molecules were eluted using the same buffer containing 1 M NaCl.

(2) HA molecule proteinase treatment The HA molecule (2.6 μg) prepared in Example 3- (1) was treated with N
4 pm each in ethyl morpholine buffer (pH 7.5)
ol lysyl endopeptidase (manufactured by Wako Pure Chemical Industries),
Digestion was performed at 37 ° C. for 1 hour using V8 protease (manufactured by Sigma) and chymotrypsin (manufactured by Boehringer). HA molecule prepared in Example 3- (1) (2.6 μg)
Was denatured at 42 ° C. for 1 hour in the presence of 2M urea. Next, 4 mM of lysyl endopeptidase, V8 protease, chymotrypsin, subtilisin (manufactured by Boehringer), proteinase K (manufactured by Boehringer), pronase (manufactured by Boehringer), and 4 mM of thermolysin (manufactured by Boehringer) were used. After digesting at 37 ° C for 12 hours in (pH 7.6),
Dialysis was performed against PBS. A part of the digested solution was taken, and the digested fragment was analyzed by dot blotting using C179 and SDS-polyacrylamide gel electrophoresis. The dot blot method was performed as follows. Place 1μ of digestive juice on a nitrocellulose filter (manufactured by MSI).
1 was loaded and dried. The same operation was repeated 5 times, and after loading a total of 5 μl of digestive juice, blocking was performed using Block Ace (Yukishiro). Next, these were reacted with a 500-fold diluted solution of a C179 solution at room temperature for 1 hour. The plate was washed with a Tris-HCl buffer (pH 7.6) containing 0.02% Tween 20, and further washed three times with a Tris-HCl buffer (pH 7.6) for 10 minutes. Further, these were added to 50 parts of an alkaline phosphatase-labeled goat anti-mouse immunoglobulin G solution (manufactured by Orgenix).
The mixture was reacted with a 0-fold diluted solution at room temperature for 1 hour, and washed similarly. Finally, the alkaline phosphatase reaction with 5-bromo-4-chloro-3-indolyl phosphate, carbonate nitroblue tetrazolium - using sodium carbonate solution (pH 9.0), was carried out in the presence of 1 mM MgCl ₂ . As a result, most of the HA molecules were undigested in the absence of urea, regardless of which protease was used. When the HA molecule denatured by urea is used as a substrate,
It was not digested with V8 protease, thermolysin or pronase. Lysyl endopeptidase, chymotrypsin and subtilisin were excessively digested, and C179
Antigenicity was completely abolished. On the other hand, when proteinase K was used, the HA molecule was digested, and the generation of a fragment polypeptide capable of binding to C179 was observed.

(3) Preparation of Stem Region Polypeptide The HA molecule prepared in Example 3- (1) (250 μg / 1
400 μl) to 100 μl of 1 M Tris-HCl (pH
7.6), 500 μl of 8M urea were added respectively, and 1 hour, 4 hours
It was kept at 2 ° C. To this solution, 2000 μl of immobilized proteinase K gel was added, and the mixture was shaken for 7 hours for 37 hours.
It was kept at ° C. Next, the reaction solution obtained by centrifugation was dialyzed against PBS for 12 hours to obtain a stem region polypeptide. The preparation of the immobilized proteinase K gel was performed as follows. 4 mg of proteinase K is added to 1 ml of H ₂
Dissolved in O, adjusted to pH 5.0 using 0.1 N HCl, added with 1 ml of ECH-Sepharose (Pharmacia), and added with 1 ml of 0.2 M EDC (pH 5.0).
It was kept at 4 ° C. for 24 hours. The gel was washed three times with 10 ml of PBS to obtain an immobilized proteinase K gel.

(4) Properties of Stem Region Polypeptide Using the stem region polypeptide described in Example 3- (3) as a test sample, the antigenicity to C179 was examined by ELISA. That is, a diluted solution of the stem region polypeptide was added to a microtiter plate (Maxi Soap: manufactured by Nunc), fixed at 37 ° C. for 1 hour 30 minutes, and then blocked using Block Ace (Snow Brand). Was. Then, these were treated with two kinds of antibodies [C179 solution (10 mg / ml)
, A 500-fold dilution of a peroxidase-labeled goat anti-mouse immunoglobulin G solution (manufactured by Kappel)] for 1 hour and 30 minutes.
And washed. Finally citric acid peroxidase reaction 0.03% H ₂ O ₂ and 1mg / ml of o- phenylenediamine dihydrochloride - was carried out using a phosphoric acid solution (pH 5.2).
The amount of antigen was calculated from the absorbance at 492 nm of the reaction solution. The HA molecule described in Example 3- (1) was used as a standard substance. From the results of the ELISA method, the stem region polypeptide was H
It was revealed that it shows the same antigenicity as the A molecule. Hemagglutination activity (HA value) of the stem region polypeptide was measured as follows. U-shaped bottom 96-hole plate (Falcon 39
11: Becton Dickinson Co., Ltd.), a two-fold serial dilution of the sample solution was performed using PBS, and an equal volume of 0.5%
The chicken red blood cell suspension was added and stirred well. After reacting at room temperature for 1 hour, aggregation of red blood cells was observed. The maximum dilution ratio at which aggregation was observed was defined as the HA value. The HA value of the stem region polypeptide is 1/1 of the HA value of the HA molecule.
000 or less.

As described above, the stem region polypeptide prepared by the protease treatment has the same antigenicity as the HA molecule,
In addition, it was found that the blood agglutinating activity derived from the bulbous region was substantially eliminated. This polypeptide is likely to become an antigenic determinant, and the globular region where an antigenic mutation is liable to occur is digested, so that the H1N1 subtype,
It can be used as a vaccine that specifically recognizes the stem region of the H2N2 subtype and induces antibodies that neutralize the virus.

Example 4 Preparation of Antigen Polypeptide (1) Preparation of HA Molecule A / Kitakyushu / 159/93 prepared in Reference Example A-1
Virus particles (40 mg) in 27 ml of 5 mM Tris-HC
1 (pH 8.0), 3 ml of 20% NP-40 was added, and the mixture was kept at 37 ° C. for 30 minutes. Next, after centrifugation,
The supernatant was collected and filtered using a 0.8 μm filter unit (Mirex-PF: manufactured by Millipore). Then, the filtrate was loaded on an ion exchange membrane (memSep DEAE: manufactured by Millipore) equilibrated with 5 mM Tris-HCl (pH 8.0), and washed with the same buffer. Further, HA molecules were eluted using the same buffer containing 1 M NaCl.

(2) HA molecule proteinase treatment The HA molecule (2.6 μg) prepared in Example 4- (1) was
In N-ethylmorpholine buffer (pH 7.5), 4
pmol lysyl endopeptidase, V8 protease,
Digestion was performed using chymotrypsin at 37 ° C. for 1 hour. HA molecule prepared in Example 4- (1) (2.6 μg)
Was denatured by incubation at 42 ° C. for 1 hour in the presence of 2M urea. Next, using 4 pmol of lysyl endopeptidase, V8 protease, chymotrypsin, subtilisin, proteinase K, pronase, and thermolysin, 5
In 0 mM Tris-HCl buffer (pH 7.6) at 37 ° C, 1
After digestion for 2 hours, dialysis was performed against PBS.
A portion of these digestions was taken, and the digestion fragments were analyzed by dot blotting using AI3C and SDS-polyacrylamide gel electrophoresis. As a result, in the absence of urea, most of the HA molecules were undigested using any of the proteases. H modified with urea
When the molecule A was used as a substrate, it was not digested with V8 protease, thermolysin or pronase. Lysyl endopeptidase, chymotrypsin and subtilisin were excessively digested, and the antigenicity against AI3C was completely abolished. On the other hand, when proteinase K was used, the HA molecule was digested, and generation of a fragment polypeptide capable of binding to AI3C was observed.

(3) Preparation of Stem Region Polypeptide The HA molecule prepared in Example 4- (1) (250 μg / 1
400 μl) to 100 μl of 1 M Tris-HCl (pH
7.6), 500 μl of 8M urea were added respectively, and 1 hour, 4 hours
It was kept at 2 ° C. To this solution, 2000 μl of immobilized proteinase K gel was added, and the mixture was shaken for 7 hours for 37 hours.
It was kept at ° C. Next, the reaction solution obtained by centrifugation was dialyzed against PBS for 12 hours to obtain a stem region polypeptide.

(4) Properties of Stem Region Polypeptide Using the stem region polypeptide described in Example 4- (3) as a test sample, the antigenicity against AI3C was examined by ELISA. That is, a diluted solution of the stem region polypeptide was added to a microtiter plate (Maxi Soap: manufactured by Nunc), fixed at 37 ° C. for 1 hour 30 minutes, and then blocked using Block Ace (Snow Brand). Was. Subsequently, these were treated with two kinds of antibodies [AI3C solution (10 mg / ml)
, A 500-fold dilution of a peroxidase-labeled goat anti-mouse immunoglobulin G solution (manufactured by Kappel)] for 1 hour and 30 minutes.
And washed. Finally citric acid peroxidase reaction 0.03% H ₂ O ₂ and 1mg / ml of o- phenylenediamine dihydrochloride - was carried out using a phosphoric acid solution (pH 5.2).
The amount of antigen was calculated from the absorbance at 492 nm of the reaction solution. The HA molecule described in Example 4- (1) was used as a standard substance. From the results of the ELISA method, the stem region polypeptide was H
It was revealed that it shows the same antigenicity as the A molecule. Hemagglutination activity (HA value) of the stem region polypeptide was measured as follows. U-shaped bottom 96-hole plate (Falcon 39
11: Becton Dickinson Co., Ltd.), a two-fold serial dilution of the sample solution was performed using PBS, and an equal volume of 0.5%
The chicken red blood cell suspension was added and stirred well. After reacting at room temperature for 1 hour, aggregation of red blood cells was observed. The maximum dilution ratio at which aggregation was observed was defined as the HA value. The HA value of the stem region polypeptide is 1/1 of the HA value of the HA molecule.
000 or less.

As described above, the stem region polypeptide prepared by the protease treatment has the same antigenicity as the HA molecule,
In addition, it was found that the blood agglutinating activity derived from the bulbous region was substantially eliminated. Since the globular region of the polypeptide, which is likely to be an antigenic determinant and is prone to antigenic mutation, is digested, it can be used as a vaccine for inducing an antibody that specifically recognizes the stem region of the H3N2 subtype. .

Example 5 Preventive Effect on Influenza Virus Escherishia coli JM109 / pENH2dH01 (FERM BP
-4190), the H of A / Okuda / 57 (H2N2)
A plasmid pENH2dH01 containing a gene encoding a polypeptide in which the globular region of the A molecule was deleted was prepared.
After treating about 5 × 10 ⁶ CV-1 cells with trypsin,
1 in 20 ml of 10% FCS-MEM (Dulbecco)
Wash twice with PBS and suspend in 1 ml of PBS. 0.8 ml thereof was placed in a cuvette for Gene Pulser, a gene transfer device from Bio-Rad, and the plasmid pENH2dH0 was added.
30 μg of 1 was added, stirred with a Pasteur pipette, and allowed to stand at 0 ° C. for 10 minutes. Set the cuvette in the gene transfer device Gene Pulser, 250 V, 960 μF
After treatment under the conditions of D, the mixture was allowed to stand at 0 ° C. for 10 minutes, and CV-
One cell was transformed. Next, the transformed cells were
Put in 0 ml of 10% FCS-MEM, 5 ml each, 6 cm in diameter
Was cultured in a Petri dish. On the third day of the culture, the expression of the polypeptide was confirmed by a staining test using C179. The cells expressing the polypeptide were treated with PBS containing trypsin, and then the cells were collected by centrifugation. Next, the collected cells were suspended in PBS, and 1 × 10 ⁵ mice were intraperitoneally administered to 10 4-week-old female BALB / c mice as vaccines. Two weeks later, a second immunization was performed in the same manner. As a control, CV-1 cells not transformed with pENH2dH01 were used, and 1 × 10 ⁵ cells were similarly transferred to 10 mice each.
The dose was administered intraperitoneally. Al / FM one week after final immunization
/ 1/47 (H1N1) per mouse at 25 μl (8
× 10 ⁴ FFU) were intranasally inoculated, and thereafter the daily survival of the mice was examined. FIG. 4 shows the results. Immunized with CV-1 cells expressing the antigenic polypeptide as shown in FIG.
In the group of mice to which the vaccine was administered (indicated by black circles in FIG. 4), 7 out of 10 mice survived on day 15 after inoculation with the virulent strain Al / FM / 1/47. On the other hand, 9 out of 10 mice died in the control mouse group (indicated by a black triangle in FIG. 4). FIG. 4 is a diagram showing the survival rates of the group to which the antigen polypeptide was administered and the group to which the antigen polypeptide was not administered. The vertical axis represents the survival rate, and the horizontal axis represents the number of days after virus infection.

As described above, it has been found that an antigen polypeptide in which the globular region of the HA molecule has been deleted can serve as a vaccine against the H1N1 subtype virus even if it is derived from the H2N2 subtype. This antigenic polypeptide is likely to be an antigenic determinant and lacks the globular region that is prone to antigenic mutations.
It can be used as a vaccine that specifically recognizes the stem region of the H2N2 subtype and induces antibodies that neutralize the virus.

Example 6 Preventive Effect on Influenza Virus Using the stem region polypeptide described in Example 3 as a test sample, the protective effect against influenza virus was examined. The stem region polypeptide was suspended in PBS and administered intraperitoneally to 4-week-old female BALB / c mice at 10 μg / 0.5 ml. The same amount was administered intraperitoneally at weekly intervals for a total of 3
Immunizations were performed. The control group received only PBS.
10 days after the last immunization, A1 / FM / 1/47 (H1N
1) 25 μl of virus (2.0 × 10 ³ ) per mouse
FFU) were inoculated intranasally, and the survival of the mice and the change in body weight of the surviving mice were observed. As shown in FIG. 5, the average body weight reduction rate of the mice immunized with the stem region polypeptide was significantly lower than that of the control group. Also, as shown in FIG. 6, 5 of 11 mice in the control group died within 7 days after virus inoculation, but 8 of 10 mice immunized with stem region polypeptide had 14 The virus survived 14 days after virus inoculation, and the survival rate 14 days after virus inoculation was 80%. On the other hand, the survival rate of the control group 14 days after the virus inoculation was 55%. FIG. 5 is a diagram showing changes in body weight of the stem region polypeptide administration group and the control group. The vertical axis indicates the average body weight of mice surviving each group, and the horizontal axis indicates the number of days after virus inoculation. FIG. 6 is a diagram showing the survival rates of the stem region polypeptide administration group and the control group, in which the vertical axis represents the survival rate and the horizontal axis represents the number of days after virus inoculation. As described above, it was found that the stem region polypeptide can be a vaccine against influenza virus.

[0123]

Industrial Applicability According to the present invention, there is provided an immunogenic polypeptide capable of producing an antibody that specifically binds to a stem region of a human influenza A virus subtype HA molecule, and a gene encoding the polypeptide. . The polypeptide can be supplied in a large amount in terms of genetic engineering and enzymology, and is not particularly affected by antigenic changes caused by changes in the spherical region of the HA molecule, and thus is particularly useful as a vaccine for preventing influenza.

[0124]

[Sequence list]

SEQ ID NO: 1 Sequence length: 5 Sequence type: number of amino acid chains: single chain Topology: linear Sequence type: peptide Fragment type: middle fragment

SEQ ID NO: 2 Sequence length: 12 Sequence type: number of amino acid chains: single chain Topology: linear Sequence type: peptide Fragment type: middle fragment

SEQ ID NO: 3 Sequence length: 5 Sequence type: number of amino acid chains: single chain Topology: linear Sequence type: peptide Fragment type: middle fragment

SEQ ID NO: 4 Sequence length: 12 Sequence type: number of amino acid chains: single-chain Topology: linear Sequence type: peptide Fragment type: middle fragment Sequence characteristics: ninth Xaa is Val or Leu.

SEQ ID NO: 5 Sequence length: 19 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid (synthetic DNA) Sequence AGCAAAAGCA GGGGATAAT 19

SEQ ID NO: 6 Sequence length: 21 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid (synthetic DNA) Sequence AGTAGAAACA AGGGTGTTTT T 21

SEQ ID NO: 7 Sequence length: 23 Sequence type: nucleic acid Number of strands: single strand Topology: linear Sequence type: other nucleic acid (synthetic DNA) Sequence TCTTTTCGAG TACTGTGTCA ACA 23

SEQ ID NO: 8 Sequence length: 23 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid (synthetic DNA) Sequence GCCCCACTAC AATTGGGGAA ATG 23

SEQ ID NO: 9 Sequence length: 24 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid (synthetic DNA) Sequence TTTTACAGAA ATTTGCTATG GCTG 24

SEQ ID NO: 10 Sequence length: 24 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid (synthetic DNA) Sequence ACTCCCCTAT TGTGACTGGG TGTA 24

SEQ ID NO: 11 Sequence length: 22 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid (synthetic DNA) Sequence GGTTATCATC ATCAGAATGA AC 22

SEQ ID NO: 12 Sequence length: 24 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid (synthetic DNA) Sequence AGTTCACCTT GTTTGTAATC CCGT 24

SEQ ID NO: 13 Sequence length: 24 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid (synthetic DNA) Sequence CCATTTTTTA CTCTTTCCAT GCAT 24

SEQ ID NO: 14 Sequence length: 24 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid (synthetic DNA) Sequence ATCTACTCAA CTGTCGCCAG TTCA 24

SEQ ID NO: 15 Sequence length: 23 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid (synthetic DNA) Sequence TTGTGTCGAC CTTCTCTGTG GAA 23

SEQ ID NO: 16 Sequence length: 20 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid (synthetic DNA) Sequence TGTAGCATTG CCGGATGGCT 20

SEQ ID NO: 17 Sequence length: 23 Sequence type: nucleic acid Number of strands: single stranded Topology: linear Sequence type: other nucleic acid (synthetic DNA) Sequence ATTATCCGGT TGCCAAAGGA TCG 23

SEQ ID NO: 18 Sequence length: 23 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid (synthetic DNA) Sequence GAGAGCACTG GTAATCTGTT GCA 23

SEQ ID NO: 19 Sequence length: 23 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid (synthetic DNA) Sequence CCATCAAATG CCTTTTGAGT GGA 23

SEQ ID NO: 20 Sequence length: 23 Sequence type: nucleic acid Number of strands: single strand Topology: linear Sequence type: other nucleic acid (synthetic DNA) Sequence ACTAGAAGCT CAGCATTGTA TGT 23

SEQ ID NO: 21 Sequence length: 24 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid (synthetic DNA) Sequence CATGCATTCA TCATCACATT TGTG 24

SEQ ID NO: 22 Sequence length: 23 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid (synthetic DNA) Sequence CATACTTGGG ATAATCATAC GTC 23

SEQ ID NO: 23 Sequence length: 23 Sequence type: nucleic acid Number of strands: single strand Topology: linear Sequence type: other nucleic acid (synthetic DNA) Sequence GCCATTTATG CTACAGTAGC AGG 23

SEQ ID NO: 24 Sequence length: 24 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid (synthetic DNA) Sequence GATCAGATTG AAGTGACTAA TGCT 24

SEQ ID NO: 25 Sequence length: 24 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid (synthetic DNA) Sequence GAATGCATCA CTCCAAATGG AAGC 24

SEQ ID NO: 26 Sequence length: 23 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid (synthetic DNA) Sequence AGGTCCTGAA TTCTCCCTTC TAC 23

SEQ ID NO: 27 Sequence length: 1754 Sequence type: nucleic acid Number of strands: double-stranded Topology: linear Sequence type: cDNA to genomic RNA Origin: Organism: A / Suita / 1/89 SEQ GGATAATAAA TACAACCAAA ATGAAAGCAA AACTACTAGT CCTGTTATGT GCATTTACAG 60 CTACAGATGC AGACACAATA TGTATAGGCT ACCATGCGAA CAACTCAACC GACACTGTTG 120 ACACAGTACT TGAGAAGAAC GTGACAGTGA CACACTCTGT CAACCTACTT GAGGACAGTC 180 ACAACGGAAA ACTATGTCGA CTAAAAGGAA TAGCCCCACT ACAATTGGGT AATTGCAGCA 240 TTGCCGGATG GATCTTAGGA AACCCAGAAT GCGAATCACT GTTTTCTAAG GAATCATGGT 300 CCTACATTGC AGAAACACCA AACTCCGAGA ATGGAACATG TTACCCAGGG TATTTCGCCG 360 ACTATGAGGA ACTGAGGGAG CAATTGAGTT CAGTATCATC ATTCGAGAGA TTCGAAATAT 420 TCCCCAAAGA AAGCTCATGG CCCAACCACA CCGTAACCAA AGGAGTAACG GCATCATGCT 480 CCCATAATGG GAAAAGCAGT TTTTACAGAA ATTTGCTATG GCTGACGGGG AAGAATGGCT 540 TGTACCCAAA TCTGAGCAAG TCCTATGA ACAACAGA GAAAGAAGTC CTTGTACTCATC GG GG T TCACATTATA GCAGGAGATT CACCCCAGAA ATAGCAAAAA 720 GACCCAAAGT AAGAGGTCAA GAAGGAAGAA TTAACTACTA CTGGACTCTG CTGGAACCCG 780 GGGACACAAT AATATTTGAG GCAAATGGAA ATCTAATAGC GCCATGGTAT GCTTTCGCAC 840 TGAGTAGAGG CTTTGGGTCA GGAATCATCA CCTCAAACGC ATCAATGGAT GAATGTGACG 900 CGAAGTGTCA AACACCCCAG GGAGCTATAA ACAGTAGTCT TCCTTTCCAG AATGTACACC 960 CAGTCACAAT AGGAGAGTGT CCAAAGTATG TCAGGAGTAC AAAATTAAGG ATGGTTACAG 1020 GACTAAGGAA CATCCCATCC ATTCAATCCA GAGGTTTGTT TGGAGCCATT GCCGGTTTCA 1080 TTGAAGGGGG GTGGACTGGA ATGATAGATG GATGGTATGG TTATCATCAT CAGAATGAAC 1140 AAGGATCTGG CTATGCTGCG GATCAAAAAA GCACACAAAA TGCCATTAAC GGAATTACAA 1200 ACAAGGTGAA TTCTGTAATC GAGAAAATGA ACACTCAATT CACAGCTGTG GGCAAAGAAT 1260 TCAACAAATT AGAAAGAAGG ATGGAATACT TAAATAAAAA AGTTGATGAT GGATTTCTGG 1320 ACATTTGGAC ATATAATGCA GAATTGTTGG TTCTACTGGA AAATGAAAGG ACTTTGGATT 1380 TTCATGACTC AAATGTGAAG AATCTGTATG AGAAAGTAAA AAGCCAATTA AAGAATAATG 1440 CCAAAGAAAT AGGATACGGG TGTTTTGAAT TCTACCACAA GTGTAACAAT GAATGCATGG 1500 AAAGTGTGAA AAATGGAACT TATGACTAT C CAAAATATTC CGAGGAATCA AAGTTAAACA 1560 GGGAAAAAAT TGATGGAGTG AAATTGGAAT CAATGGGAGT CTATCAGATT CTGGCGATCT 1620 ACTCAACTGT CGCCAGTTCA CTGGTGCTTT TGGTCTAACGAGCTTCTGGATCAAG TAGTGTTCTAATG

SEQ ID NO: 28 Sequence length: 1728 Sequence type: nucleic acid Number of strands: double-stranded Topology: linear Sequence type: cDNA to genomic RNA Origin: Organism: A / Izumi / 5/65 SEQ ATAGACAACC AAAAGCATAA CAATGGCCAT CATCTATCTC ATACTCCTGT TCACAGCAGT 60 GAGGGGGGAC CAGATATGCA TTGGATACCA TGCCAATAAT TCCACAGAAA AGGTCGACAC 120 AATTCTAGAG CGGAATGTCA CTGTGACTCA TGCCAAGGAC ATCCTTGAGA AGACCCACAA 180 CGGAAAGCTA TGCAAACTAA ACGGAATCCC TCCACTTGAA CTAGGGGACT GTAGCATTGC 240 CGGATGGCTC CTTGGAAATC CAGAATGTGA TAGGCTTCTA AGGGTGCCAG AATGGTCCTA 300 TATAATGGAG AAAGAAAACC CGAGATACAG TTTATGTTAC CCAGGCAACT TCAATGACTA 360 TGAAGAATTG AAACATCTCC TCAGCAGCGT AAAACATTTC GAGAAAGTAA AGATTCTGCC 420 CAAAGATAGA TGGACACAGC ATACAACAAC TGGAGGTTCA AAGGCCTGCG CAGTGTCAGG 480 TAAACCATCA TTCTTCAGGA ACATGGTCTG GCTGACAAAG AAAGGACCAA ATTATCCGGT 540 TGCCAAAGGA TCGTACAACA ATACGAGCCAGA TGAGCAG AGAG CATCGATCGATCGATCAGGATCAGGATCAGGATCAGGATCAGGATGCAGG A ACAAAAGGTC AATCCCTGAA ATAGCAGCAA GGCCTAAAGT 720 GAATGGACTA GGAAGTAGAA TGGAATTCTC TTGGACCCTC TTGGATGTGT GGGACACCAT 780 AAATTTTGAG AGCACTGGTA ATCTAGTTGC ACCAGAGTAT GGATTCAAAA TATCGAAAAG 840 AGGTAGTTCA GGGATCATGA AGACAGAAGG AACACTTGGG AACTGTGAGA CCAAATGCCA 900 AACTCCTTTG GGAGCAATAA ATACAACACT ACCTTTTCAC AATGTCCACC CACTGACAAT 960 AGGTGAATGC CCCAAATATG TAAAATCGGA GAAATTGGTC TTAGCAACAG GACTAAGGAA 1020 TGTTCCCCAG ATTGAATCAA GAGGATTGTT TGGGGCAATA GCTGGCTTTA TAGAAGGAGG 1080 ATGGCAAGGA ATGGTTGATG GTTGGTATGG ATACCATCAC AGCAATGACC AGGGATCAGG 1140 GTATGCAGCA GACAAAGAAT CCACTCAAAA GGCATTTGAT GGAATCACCA ACAAGGTAAA 1200 TTCTGTGATT GAAAAGATGA ACACCCAATT TGAAGCTGTT GGGAAAGAAT TCAATAATTT 1260 AGAGAAAAGA CTGGAGAACT TGAACAAAAA GATGGAAGAC GGGTTTCTAG ATGTGTGGAC 1320 ATACAATGCT GAGCTTCTAG TTCTGATGGA AAATGAGAGG ACACTTGACT TCCATGATTC 1380 TAATGTCAAG AACCTGTATG ATAAAGTCAG AATGCAGCTG AGAGACAACG TCAAAGAACT 1440 AGGAAATGGA TGTTTTGAAT TTTATCACAA ATGTGACGAT GAATGCATGA ATAGTGTGAA 1500 AAACGGGACG TATGATTATC CCAAGTATG A AGAAGAATCT AAACTAAATA GAAATGAAAT 1560 CAAAGGGGTA AAATTGAGCA GCATGGGGGT TTACCAAATT CTTGCCATTT ATGCTACAGT 1620 TGCAGGTTCT CTGTCACTGG CAATCATGAT GGCTGGGATC TCTTTCTGGA TGTGCTCCAA 1680 CGGGTCTCTATGAGGTGATGTATGATGTGATAGTATG

SEQ ID NO: 29 Sequence length: 442 Sequence type: nucleic acid Number of strands: double-stranded Topology: linear Sequence type: cDNA to genomic RNA Origin: Organism: A / PR / 8/34 SEQ CCTTTCCAGA ATATACACCC AGTCACAATA GGAGAGTGCC CAAAATACGT CAGGAGTGCC 60 AAATTGAGGA TGGTTACAGG ACTAAGGAAC ATCCCGTCCA TTCAATCCAG AGGTCTATTT 120 GGAGCCATTG CCGGTTTTAT TGAAGGGGGA TGGACTGGAA TGATAGATGG ATGGTATGGT 180 TATCATCATC AGAATGAACA GGGATCAGGC TATGCAGCGG ATCAAAAAAG CACACAAAAT 240 GCCATTAACG GGATTACAAA CAAGGTGAAC TCTGTTATCG AGAAAATGAA CACTCAATTC 300 ACAGCTGTGG GTAAAGAATT CAACAAATTA GAAAAAAGGA TGGAAAATTT AAATAAAAAA 360 GTTGATGATG GATTTCTGGA CATTTGGACA TATAATGCAG AATTGTTAGT TCTACTGGAA 420 AATGAAAGGA CTCTGGATTT CC 442

SEQ ID NO: 30 Sequence length: 424 Sequence type: nucleic acid Number of strands: double-stranded Topology: linear Sequence type: cDNA to genomic RNA Origin: Organism: A / Bangkok / 10/83 SEQ CCTTTCCAGA ATGTACACCC AGTCACAATA GGAGAGTGCC CAAAGTACGT CAGGAGTACA 60 AAATTAAGGA TGGTTACAGG ACTAAGGAAC ATCCCATCCA TTCAATCCAG AGGTTTGTTT 120 GGAGCCATTG CCGGTTTCAT TGAAGGGGGA TGGACTGGAA TGATAGATGG ATGGTATCGT 180 TATCATCATC AGAATGAACA AGGATCTGGC TATGCTGCGG ATCAAAAAAG CACACAAAAT 240 GCCATTAACG GGATTACAAA CAAGGTGAAC TCTGTAATCG AGAAAATGAA CACTCAATTC 300 ACAGCTGTGG GTAAAGAATT CAACAAATTA GAAAAAAGGA TGGAAAACTT AAATAAAAAA 360 GTTGATGATG GATTTCTGGA CATTTGGACA TATAATGCAG AATTGTTGGT TCTACTGGAA 420 AATG 424

SEQ ID NO: 31 Sequence length: 424 Sequence type: nucleic acid Number of strands: double-stranded Topology: linear Sequence type: cDNA to genomic RNA Origin: Organism: A / Yamagata / 120/86 SEQ CCTTTCCAGA ATGTACACCC AGTCACAATA GGAGAGTGCC CAAAGTATGT CAGGAGTACA 60 AAATTAAGGA TGGTTACAGG ACTAAGGAAC ATCCCATCCA TTCAATCCAG AGGTTTGTTT 120 GGAGCCATTG CCGGTTTCAT TGAAGGGGGG TGGACTGGAA TGATAGATGG ATGGTATGGT 180 TATCATCATC AGAATGAACA AGGATCTGGC TATGCTGCGG ATCAAAAAAG CACACAAAAT 240 GCCATTAACG GGATTACAAA CAAGGTGAAT TCTGTAATCG AGAAAATGAA CACTCAATTC 300 ACAGCTGTGG GCAAAGAATT CAACAAATTA GAAAGAAGGA TGGAAAACTT AAATAAAAAA 360 GTTGATGATG GATTTCTGGA CATTTGGACA TATAATGCAG AATTGTTGGT CCTACTGGAA 420 AATG 424

SEQ ID NO: 32 Sequence length: 429 Sequence type: nucleic acid Number of strands: double-stranded Topology: linear Sequence type: cDNA to genomic RNA Origin: Organism: A / Osaka / 930/88 SEQ CCTTTCCAGA ATGTACACCC AGTCACAATA GGAGAGTGCC CAAAGTATGT CAGGAGTACA 60 AAATTAAGGA TGGTTACAGG ACTAAGGAAC ATCCCATCCA TTCAATCCAG AGGTTTGTTT 120 GGAGCCATTG CCGGTTTCAT AGAAGGGGGG TGGACTGGAA TGATAGATGG ATGGTATGGT 180 TATCATCATC AGAATGAACA AGGATCTGGC TATGCTGCGG ATCAAAAAAG CACACAAAAT 240 GCCATTAACG GAATTACAAA CAAGGTGAAT TCTGTAATCG AGAAAATGAA CACTCAATTC 300 ACAGCTGTGG GCAAAGAATT CAACAAATTA GAAAGAAGGA TGGAAAACTT AAATAAAAAA 360 GTTGATGATG GATTTCTGGA CATTTGGACA TATAATGCAG AATTGTTGGT TCTACTGGAA 420 AATGAAAGG 429

SEQ ID NO: 33 Sequence length: 400 Sequence type: nucleic acid Number of strands: double-stranded Topology: linear Sequence type: cDNA to genomic RNA Origin: Organism: A / Okuda / 57 sequence GCAATAAATA CAACATTACC TTTTCACAAT GTCCACCCAC TGACAATAGG TGAGTGCCCC 60 AAATATGTAA AATCGGAGAA GTTGGTCTTA GCAACAGGAC TAAGGAATGT TCCCCAGATT 120 GAATCAAGAG GATTGTTTGG GGCAATAGCT GGTTTTATAG AAGGAGGATG GCAAGGAATG 180 GTTGACGGTT GGTATGGATA CCATCACAGC AATGACCAGG GATCAGGGTA TGCAGCAGAC 240 AAAGAATCCA CTCAAAAGGC ATTTGATGGA ATCACCAACA AGGTAAATTC TGTGATTGAA 300 AAGATAAACA CCCAATTTGA AGCTGTTGGG AAAGAATTCG GTAACTTAGA GAAAAGACTG 360 GAGAACTTGA ACAAAAAGAT GGAAGACGGG TTTCTAGATG 400

SEQ ID NO: 34 Sequence length: 409 Sequence type: Nucleic acid Number of strands: Double-stranded Topology: Linear Sequence type: cDNA to genomic RNA Origin: Organism name: A / Adachi / 2/57 SEQ CGCCTTGGAG CAATAAATAC AACATTGCCT TTTCACAATG TCCACCCACT GACAATAGGT 60 GAGTGCCCCA AATATGTAAA ATCGGAGAAG TTGGTCTTAG CAACAGGACT AAGGAATGTT 120 CCCCAGATTG AATCAAGAGG ATTGTTTGGG GCAATAGCTG GTTTTATAGA AGGAGGATGG 180 CAAGGAATGG TTGATGGTTG GTATGGATAC CATCACAGCA ATGACCAGGG ATCAGGGTAT 240 GCAGCAGACA AAGAATCCAC TCAAAAGGCA TTTGATGGAA TCACCAACAA GGTAAATTCT 300 GTGATTGAAA AGATGAACAC CCAATTTGAA GCTGTTGGGA AAGAATTCGG TAACTTAGAG 360 AGAAGACTGG AGAACTTGAA CAAAAAGATG GAAGACGGGT TTCTAGATG 409

SEQ ID NO: 35 Sequence length: 410 Sequence type: nucleic acid Number of strands: double-stranded Topology: linear Sequence type: cDNA to genomic RNA Origin: Organism name: A / Kumamoto / 1/65 SEQ CTCCTTTGGA GCAATAAATA CAACATTACC TTTTCACAAT GTCCACCCAC TGACAATAGG 60 TGAATGCCCC AAATATGTAA AATCGGAGAA ACTGGTCTTA GCAACAGGAC TAAGGAATGT 120 TCCCCAGATT GAATCAAGAG GATTGTTTGG GGCAATAGCT GGCTTTGTAG AAGGAGGATG 180 GCAAGGAATG ATTGATGGTT GGTATGGATA CCATCACAGC AATGATCAGG GATCAGGGTT 240 TGCAGCAGAC AAAGAATCCA CTCAAAAGGC ATTTGATGGA ATCACCAACA AGGTAAATTC 300 TGTGATTGAA AAGATGAACA CCCAATTTGA AGCTGTTGGG AAAGAATTCA ATAATTTAGA 360 GAAAAGACTG GAGAACTTGA ACAAAAGGAT GGAAGACGGG TTTCTAGATG 410

SEQ ID NO: 36 Sequence length: 394 Sequence type: nucleic acid Number of strands: double-stranded Topology: linear Sequence type: cDNA to genomic RNA Origin: Organism name: A / Kaizuka / 2/65 SEQ AATACAACAC TACCTTTTCA CAATGTCCAC CCACTGACAA TAGGTGAATG CCCCAAATAT 60 GTAAAATCGG AGAAATTGGT CTTAGCAACA GGACTAAGGA ATGTTCCCCA GATTGAATCA 120 AGAGGATTGT TTGGGGCAAT AGCTGGCTTT ATAGAAGGAG GATGGCAAGG AATGGTTGAT 180 GGTTGGTATG GATACCATCA CAGCAATGAC CAGGGATCAG GGTATGCAGC AGACAAAGAA 240 TCCACTCAAA AGGCATTTGA TGGAATCACC AACAAGGTAA ATTCTGTGAT TGAAAAGATG 300 AACACCCAAT TTGAAGCTGT TGGGAAAGAA TTCAATAATT TAGAGAAAAG ACTGGAGAAC 360 TTGAACAAAA AGATGGAAGA CGGGTTTCTA GATG 394

SEQ ID NO: 37 Sequence length: 329 Sequence type: nucleic acid Number of strands: double-stranded Topology: linear Sequence type: cDNA to genomic RNA Origin: Organism: A2 / Aichi / 2/68 SEQ ATGACAAGCC CTTTCAAAAC GTAAACAAGA TCACATATGG AGCATGCCCC AAGTATGTTA 60 AGCAAAACAC CCTGAAGTTG GCAACAGGGA TGCGGAATGT ACCAGAGAAA CAAACTAGAG 120 GCCTATTCGG CGCAATAGCA GGTTTCATAG AAAATGGTTG GGAGGGAATG ATAGACGGTT 180 GGTACGGTTT CAGGCATCAA AATTCTGAGG GCACAGGACA AGCAGCAGAT CTTAAAAGCA 240 CTCAAGCAGC CATCGACCAA ATCAATGGGA AATTGAACAG GGTAATCGAG AAGACGAACG 300 AGAAATTCCA TCAAATCGAA AAGGAATTC 329

SEQ ID NO: 38 Sequence length: 334 Sequence type: Nucleic acid Number of strands: Double-stranded Topology: Linear Sequence type: cDNA to genomic RNA Origin: Organism: A / Fukuoka / C29 / 85 SEQ ATGACAAACC CTTTCAAAAT GTAAACAAGA TCACATATGG GGCATGTCCC AGGTATGTTA 60 AGCAAAACAC TCTGAAATTG GCAACAGGGA TGCGGAATGT ACCAGAGAAA CAAACTAGAG 120 GCATATTCGG CGCAATAGCA GGTTTCATAG AAAATGGTTG GGAGGGAATG GTAGACGGTT 180 GGTACGGTTT CAGGCATCAA AATTCTGAGG GCACAGGACA AGCAGCAGAT CTTAAAAGCA 240 CTCAAGCAGC AATCGACCAA ATCAACGGGA AACTGAATAG GTTAATCGAG AAGACGAACG 300 AGAAATTCCA TCAAATCGAA AAGGAATTCT CAGA 334

SEQ ID NO: 39 Sequence length: 329 Sequence type: nucleic acid Number of strands: double-stranded Topology: linear Sequence type: cDNA to genomic RNA Origin: Organism name: A / Sichuan / 2/87 SEQ ATGACAAACC CTTTCAAAAT GTAAACAAGA TCACATATGG GGCATGTCCC AGATATGTTA 60 AGCAAAACAC TCTGAAATTG GCAACAGGGA TGCGGAATGT ACCAGAGAAA CAAACTAGAG 120 GCATATTCGG CGCAATAGCA GGTTTCATAG AAAATGGTTG GGAGGGAATG GTAGACGGCT 180 GGTACGGTTT CAGGCATCAA AATTCTGAGG GCACAGGACA AGCAGCAGAT CTTAAAAGCA 240 CTCAAGCAGC AATCGACCAA ATCAACGGGA AACTGAATAG GTTAATCGAG AAGACGAACG 300 AGAAATTCCA TCAAACCGAA AAGGAATTC 329

SEQ ID NO: 40 Sequence length: 334 Sequence type: Nucleic acid Number of strands: Double-stranded Topology: Linear Sequence type: cDNA to genomic RNA Origin: Organism name: A / Ibaraki / 1/90 SEQ ATGACAAACC CTTTCAAAAT ATAAACAGGA TCACATATGG GGCATGTCCC AGATATGTTA 60 AGCAAAACAC TCTGAAATTG GCAACAGGGA TGCGGAATGT ACCAGAGAAA CAAACTAGAG 120 GCATATTCGG CGCAATCGCA GGTTTCATAG AAAATGGTTG GGAGGGAATG GTAGACGGTT 180 GGTACGGTTT CAGGCATCAA AATTCTGAGG GCACAGGACA AGCAGCAGAT CTTAAAAGCA 240 CTCAAGCAGC AATCGACCAA ATCAACGGGA AACTGAATAG GTTAATCGAG AAGACGAACG 300 AGAAATTCCA TCAAATCGAA AAGGAATTCT CAGA 334

SEQ ID NO: 41 Sequence length: 329 Sequence type: nucleic acid Number of strands: double-stranded Topology: linear Sequence type: cDNA to genomic RNA Origin: Organism name: A / Suita / 1/90 SEQ ATGACAAACC CTTTCAAAAT GTAAACAGGA TCACATATGG GGCATGTCCC AGATATGTTA 60 AGCAAAACAC TCTGAAATTG GCAACAGGGA TGCGGAATGT ACCAGAAAAA CAAACTAGGG 120 GCATATTCGG CGCAATCGCA GGTTTCATAG AAAATGGTTG GGAGGGAATG GTAGACGGTT 180 GGTACGGTTT CAGGCATCAA AACTCTGAGG GCACAGGACA AGCAGCAGAT CTTAAAAGCA 240 CTCAAGCAGC AATCGACCAA ATCAACGGGA AACTGAATAG GTTAATCGAG AAGACGAACG 300 AGAAATTCCA TCAAACCGAA AAGGAATTC 329

SEQ ID NO: 42 Sequence length: 30 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid (synthetic DNA) Sequence GATCTAGAAG CAAAAGCAGG GGTTATACCA 30

SEQ ID NO: 43 Sequence length: 30 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid (synthetic DNA) Sequence CGGCTAGCAA AAGCAGGGGT TATACCATAG 30

SEQ ID NO: 44 Sequence length: 29 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid (synthetic DNA) Sequence ACAGATCTAG TAGAAACAAG GGTGTTTTT 29

SEQ ID NO: 45 Sequence length: 30 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid (synthetic DNA) Sequence CGGCTAGCAG AAACAAGGGT GTTTTTAATT 30

SEQ ID NO: 46 Sequence length: 1783 Sequence type: Nucleic acid Number of strands: Double-stranded Topology: Linear Sequence type: cDNA to genomic RNA Origin: Organism: A / Okuda / 57 Sequence CGGCTAGCAA AAGCAGGGGT TATACCATAG AAAACCAAAA 40 GCAAAACA ATG GCC ATC ATT TAT CTC ATT CTC CTG TTC ACA GCA GTG AGA GGG 93 Met Ala Ile Ile Tyr Leu Ile Leu Leu Phe Thr Ala Val Arg Gly -15 -10 -5 GAC CAG ATA TGC ATT GTAC GCC AAT AAT TCC ACA GAG AAG 138 Asp Gln Ile Cys Ile Gly Tyr His Ala Asn Asn Ser Thr Glu Lys 1 5 10 15 GTC GAC ACA ATT CTA GAG CGG AAC GTC ACT GTG ACT CAT GCC AAG 183 Val Asp Thr Ile Leu Glu Arg Asn Val Thr Val Thr His Ala Lys 20 25 30 GAC ATC CTT GAG AAG ACC CAT AAC GGA AAG TTA TGC AAA CTA AAC 228 Asp Ile Leu Glu Lys Thr His Asn Gly Lys Leu Cys Lys Leu Asn 35 40 45 GGA ATC CCT CCA CTT GAA CTA GGG GAC TGT AGC ATT GCC GGA TGG 273 Gly Ile Pro Pro Leu Glu Leu Gly Asp Cys Ser Ile Ala Gly Trp 50 55 60 CTC CTT GGA AAT CCA AAA TGT GAT AGG CTT CTA AGT GTG CCA GAA 318 Leu Leu Gly Asn Pro Lys Cys Asp Arg Leu Leu Ser Val Pro Glu 65 70 75 CGG TCC TAT ATA TTG GAG AAA GAA AAC CCG AGA GAC GGT TTG TGT 363 Arg Ser Tyr Ile Leu Glu Lys Glu Asn Pro Arg Asp Gly Leu Cys 80 85 90 TAT CCA GGC AGC TTC AAT GAT TAT GAA GAA TTG AAA CAT CTC CTC 408 Tyr Pro Gly Ser Phe Asn Asp Tyr Glu Glu Leu Lys His Leu Leu 95 100 105 AGC AGC GTG AAA CAT TTC GAG AAA GTA AAG ATT CTG CCC AAA GAT 453 Ser Ser Val Lys His Phe Glu Lys Val Lys Ile Leu Pro Lys Asp 110 115 120 AGA TGG ACA CAG CAT ACA ACA ACT GGA GGT TCA CGG GCC TGC GCG 498 Arg Trp Thr Gln His Thr Thr Thr Gly Gly Ser Arg Ala Cys Ala 125 130 135 GTG TCT GGT AAT CCA TCA TTT TTC AGG AAC ATG GTC TGG CTG ACA 543 Val Ser Gly Asn Pro Ser Phe Phe Arg Asn Met Val Trp Leu Thr 140 145 150 AAG GAA GGA TCA GAT TAT CCG GTT GCC AAA GGA TCG TAC AAC AAT 588 Lys Glu Gly Ser Asp Tyr Pro Val Ala Lys Gly Ser Tyr Asn Asn 155 160 165 ACA AGC GGA GAA CAA ATG CTA ATA ATT TGG GGG GTG CAC CAT CCC 633 Thr Ser Gly Glu Gln Met Leu Ile Ile Trp Gly Val His His Pr o 170 175 180 ATT GAT GAG ACA GAA CAA AGA ACA TTG TAC CAG AAT GTG GGA ACC 678 Ile Asp Glu Thr Glu Gln Arg Thr Leu Tyr Gln Asn Val Gly Thr 185 190 195 TAT GTT TCC GTA GGC ACA TCA ACA TTG AAC AAA AGG TCA ACC CCA 723 Tyr Val Ser Val Gly Thr Ser Thr Leu Asn Lys Arg Ser Thr Pro 200 205 210 GAA ATA GCA ACA AGG CCT AAA GTG AAT GGA CAA GGA GGT AGA ATG 768 Glu Ile Ala Thr Arg Pro Lys Val Asn Gly Gln Gly Gly Arg Met 215 220 225 GAA TTC TCT TGG ACC CTC TTG GAT ATG TGG GAC ACC ATA AAT TTT 813 Glu Phe Ser Trp Thr Leu Leu Asp Met Trp Asp Thr Ile Asn Phe 230 235 240 GAG AGT ACT GGT AAT CTA ATT GCA CCA GAG TAT GGA TTC AAA ATA 858 Glu Ser Thr Gly Asn Leu Ile Ala Pro Glu Tyr Gly Phe Lys Ile 245 250 255 TCG AAA AGA GGT AGT TCA GGG ATC ATG AAA ACA GAA GGA ACA CTT 903 Ser Lys Arg Gly Ser Ser Gly Ile Met Lys Thr Glu Gly Thr Leu 260 265 270 GAG AAC TGT GAG ACC AAA TGC CAA ACT CCT TTG GGA GCA ATA AAT 948 Glu Asn Cys Glu Thr Lys Cys Gln Thr Pro Leu Gly Ala Ile Asn 275 280 285 285 ACA ACA TTA CCT TTT CAC AAT GTC CA C CCA CTG ACA ATA GGT GAG 993 Thr Thr Leu Pro Phe His Asn Val His Pro Leu Thr Ile Gly Glu 290 295 300 TGC CCC AAA TAT GTA AAA TCG GAG AAG TTG GTC TTA GCA ACA GGA 1038 Cys Pro Lys Tyr Val Lys Ser Glu Lys Leu Val Leu Ala Thr Gly 305 310 315 CTA AGG AAT GTT CCC CAG ATT GAA TCA AGA GGA TTG TTT GGG GCA 1083 Leu Arg Asn Val Pro Gln Ile Glu Ser Arg Gly Leu Phe Gly Ala 320 325 330 ATA GCT GGT TTT ATA GAA GGA GGA TGG CAA GGA ATG GTT GAC GGT 1128 Ile Ala Gly Phe Ile Glu Gly Gly Trp Gln Gly Met Val Asp Gly 335 330 345 TGG TAT GGA TAC CAT CAC AGC AAT GAC CAG GGA TCA GGG TAT GCA 1173 Trp Tyr Gly Tyr His His Ser Asn Asp Gln Gly Ser Gly Tyr Ala 350 355 360 GCA GAC AAA GAA TCC ACT CAA AAG GCA TTT GAT GGA ATC ACC AAC 1218 Ala Asp Lys Glu Ser Thr Gln Lys Ala Phe Asp Gly Ile Thr Asn 365 370 375 AAG GTA AAT TCT GTG ATT GAA AAG ATA AAC ACC CAA TTT GAA GCT 1263 Lys Val Asn Ser Val Ile Glu Lys Ile Asn Thr Gln Phe Glu Ala 380 385 390 GTT GGG AAA GAA TTC GGT AAC TTA GAG AAA AGA CTG GAG AAC TTG 1308 Val Gly Lys Glu Phe Gly Asn Leu Glu Lys Arg Leu Glu Asn Leu 395 400 405 AAC AAA AAG ATG GAA GAC GGG TTT CTA GAT GTG TGG ACA TAC AAT 1353 Asn Lys Lys Met Glu Asp Gly Phe Leu Asp Val Trp Thr Tyr Asn 410 415 420 GCT GAG CTT TTA GTT CTG ATG GAA AAT GAG AGG ACA CTT GAC TTT 1398 Ala Glu Leu Leu Val Leu Met Glu Asn Glu Arg Thr Leu Asp Phe 425 430 435 CAT GAT TCT AAT GTC AAG AAT CTG TAT AGT AAA GTC AGA ATG CAG 1443 His Asp Ser Asn Val Lys Asn Leu Tyr Ser Lys Val Arg Met Gln 440 445 450 CTG AGA GAC AAC GTC AAA GAA CTA GGA AAT GGA TGT TTT GAA TTT 1488 Leu Arg Asp Asn Val Lys Glu Leu Gly Asn Gly Cys Phe Glu Phe 455 460 465 TAT CAC AAA TGT GAT GAT GAA TGC ATG AAT AGT GTG AAA AAC GGG 1533 Tyr His Lys Cys Asp Asp Glu Cys Met Asn Ser Val Lys Asn Gly 470 475 480 480 ACA TAT GAT TAT CCC AAG TAT GAA GAA GAG TCT AAA CTA AAT AGA 1578 Thr Tyr Asp Tyr Pro Lys Tyr Glu Glu Glu Ser Lys Leu Asn Arg 495 500 505 AAT GAA ATC AAA GGG GTA AAA TTG AGC AGC ATG GGG GTT TAT CAA 1623 Asn Glu Ile Lys Gly Val Lys Leu Ser Ser Met Gly Val Tyr Gln 510 515 520 ATC CTT GCC ATT TAT GCT ACA GTA GCA GGT TCT ATG TCA CTG GCA 1668 Ile Leu Ala Ile Tyr Ala Thr Val Ala Gly Ser Met Ser Leu Ala 525 530 535 535 ATC ATG ATG GCT GGG ATC TCT TTC TGG GTG TGC TCC AAC GGG TCT 1713 Ile Met Met Ala Gly Ile Ser Phe Trp Val Cys Ser Asn Gly Ser 540 545 550 CTG CAG TGC AGG ATC TGC ATA TGATTATAAG TCATTTTATA ATTAAAAACA 1764 Leu Gln Cys Arg Ile Cys Ile 555 CCCTTGTTTC TCGTAGCC

SEQ ID NO: 47 Sequence length: 25 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acids (synthetic DNA) Sequence TCCGTTTAGT TTGCATAACT TTCCG 25

SEQ ID NO: 48 Sequence length: 26 Sequence type: nucleic acid Number of strands: single strand Topology: linear Sequence type: other nucleic acid (synthetic DNA) Sequence TCCGGGATCA TGAAAACAGA AGGAAC 26

SEQ ID NO: 49 Sequence length: 1135 Sequence type: nucleic acid Number of strands: double-stranded Topology: linear Sequence type: cDNA to genomic RNA Origin: Organism: A / Okuda / 57 sequence CTAGCAAAAG CAGGGGTTAT ACCATAGAAA ACCAAAAGCA AAACAATGGC CATCATTTAT 60 CTCATTCTCC TGTTCACAGC AGTGAGAGGG GACCAGATAT GCATTGGATA CCATGCCAAT 120 AATTCCACAG AGAAGGTCGA CACAATTCTA GAGCGGAACG TCACTGTGAC TCATGCCAAG 180 GACATCCTTG AGAAGACCCA TAACGGAAAG TTATGCAAAC TAAACGGATC CGGGATCATG 240 AAAACAGAAG GAACACTTGA GAACTGTGAG ACCAAATGCC AAACTCCTTT GGGAGCAATA 300 AATACAACAT TACCTTTTCA CAATGTCCAC CCACTGACAA TAGGTGAGTG CCCCAAATAT 360 GTAAAATCGG AGAAGTTGGT CTTAGCAACA GGACTAAGGA ATGTTCCCCA GATTGAATCA 420 AGAGGATTGT TTGGGGCAAT AGCTGGTTTT ATAGAAGGAG GATGGCAAGG AATGGTTGAC 480 GGTTGGTATG GATACCATCA CAGCAATGAC CAGGGATCAG GGTATGCAGC AGACAAAGAA 540 TCCACTCAAA AGGCATTTGA TGGAATCACC AACAAGGTAA ATTCTGTGAT TGAAAAGATA 600GAACGA TGA GAGA TGA GAGA TGA GAGA TGA GAGA TGA GAGA GAGA CGGGTTTCTA GATGTGTGGA CATACAATGC TGAGCTTTTA 720 GTTCTGATGG AAAATGAGAG GACACTTGAC TTTCATGATT CTAATGTCAA GAATCTGTAT 780 AGTAAAGTCA GAATGCAGCT GAGAGACAAC GTCAAAGAAC TAGGAAATGG ATGTTTTGAA 840 TTTTATCACA AATGTGATGA TGAATGCATG AATAGTGTGA AAAACGGGAC ATATGATTAT 900 CCCAAGTATG AAGAAGAGTC TAAACTAAAT AGAAATGAAA TCAAAGGGGT AAAATTGAGC 960 AGCATGGGGG TTTATCAAAT CCTTGCCATT TATGCTACAG TAGCAGGTTC TATGTCACTG 1020 GCAATCATGA TGGCTGGGAT CTCTTTCTGG GTGTGCTCCA ACGGGTCTCT GCAGTGCAGG 1080 ATCTGCATAT GATTATAAGT CATTTTATAA TTAAAAACAC CCTTGTTTCT GCTAG 1135

SEQ ID NO: 50 Sequence length: 348 Sequence type: amino acid Number of chains: single chain Topology: linear Sequence type: peptide Sequence Met Ala Ile Ile Tyr Leu Ile Leu Leu Phe Thr Ala Val Arg Gly -15 -10 -5 Asp Gln Ile Cys Ile Gly Tyr His Ala Asn Asn Ser Thr Glu Lys 1 5 10 15 Val Asp Thr Ile Leu Glu Arg Asn Val Thr Val Thr His Ala Lys 20 25 30 Asp Ile Leu Glu Lys Thr His Asn Gly Lys Leu Cys Lys Leu Asn 35 40 45 Gly Ser Gly Ile Met Lys Thr Glu Gly Thr Leu Glu Asn Cys Glu 50 55 60 Thr Lys Cys Gln Thr Pro Leu Gly Ala Ile Asn Thr Thr Leu Pro 65 70 75 Phe His Asn Val His Pro Leu Thr Ile Gly Glu Cys Pro Lys Tyr 80 85 90 Val Lys Ser Glu Lys Leu Val Leu Ala Thr Gly Leu Arg Asn Val 95 100 105 Pro Gln Ile Glu Ser Arg Gly Leu Phe Gly Ala Ile Ala Gly Phe 110 115 120 Ile Glu Gly Gly Trp Gln Gly Met Val Asp Gly Trp Tyr Gly Tyr 125 130 135 His His Ser Asn Asp Gln Gly Ser Gly Tyr Ala Ala Asp Lys Glu 140 145 150 Ser Thr Gln Lys Ala Phe Asp Gly Ile Thr Asn Lys Val Asn Ser 155 160 165 Val Ile Glu Lys Ile Asn Thr Gln Phe Glu Ala Val Gly Lys Glu 170 175 180 Phe Gly Asn Leu Glu Lys Arg Leu Glu Asn Leu Asn Lys Lys Met 185 190 195 Glu Asp Gly Phe Leu Asp Val Trp Thr Tyr Asn Ala Glu Leu Leu 200 205 210 Val Leu Met Glu Asn Glu Arg Thr Leu Asp Phe His Asp Ser Asn 215 220 225 Val Lys Asn Leu Tyr Ser Lys Val Arg Met Gln Leu Arg Asp Asn 230 235 240 Val Lys Glu Leu Gly Asn Gly Cys Phe Glu Phe Tyr His Lys Cys 245 250 255 Asp Asp Glu Cys Met Asn Ser Val Lys Asn Gly Thr Tyr Asp Tyr 260 265 270 270 Pro Lys Tyr Glu Glu Glu Ser Lys Leu Asn Arg Asn Glu Ile Lys 275 280 285 Gly Val Lys Leu Ser Ser Met Gly Val Tyr Gln Ile Leu Ala Ile 290 295 300 Tyr Ala Thr Val Ala Gly Ser Met Ser Leu Ala Ile Met Met Ala 305 310 315 Gly Ile Ser Phe Trp Val Cys Ser Asn Gly Ser Leu Gln Cys Arg 320 325 330 Ile Cys Ile

SEQ ID NO: 51 Sequence length: 30 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid (synthetic DNA) Sequence GATCTAGAAG CAAAGCAGGG GATAATTCTA 30

SEQ ID NO: 52 Sequence length: 29 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid (synthetic DNA) Sequence ACAGATCTAG TAGAAACAAG GGTGTTTTT 29

SEQ ID NO: 53 Sequence length: 30 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid (synthetic DNA) Sequence CGGCTAGCAG AAACAAGGGT GTTTTTAATT 30

SEQ ID NO: 54 Sequence length: 1778 Sequence type: nucleic acid Number of strands: double-stranded Topology: linear Sequence type: cDNA to genomic RNA Origin: Organism name: A2 / Aichi / 2/68 Sequence GATCTAGAAG CAAAGCAGGG GATAATTCTA 30 TTAATC ATG AAG ACC ATC ATT GCT TTG AGC TAC ATT TTC TGT CTG GCT CTC 81 Met Lys Thr Ile Ile Ala Leu Ser Tyr Ile Phe Cys Leu Ala Leu -15 -10 -5 GGC CAA GAC CTCCA GAC AAC AGC ACA GCA ACG CTG TGC 127 Gly Gln Asp Leu Pro Gly Asn Asp Asn Ser Thr Ala Thr Leu Cys 1 5 10 CTG GGA CAT CAT GCG GTG CCA AAC GGA ACA CTA GTG AAA ACA ATC 172 Leu Gly His His Ala Val Pro Asn Gly Thr Leu Val Lys Thr Ile 15 20 25 ACA GAT GAT CAG ATT GAA GTG ACT AAT GCT ACT GAG CTA GTT CAG 217 Thr Asp Asp Gln Ile Glu Val Thr Asn Ala Thr Glu Leu Val Gln 30 35 40 AGC TCC TCA ACG GGG AAA ATA TGC AAC AAT CCT CAT CGA ATC CTT 262 Ser Ser Ser Thr Gly Lys Ile Cys Asn Asn Pro His Arg Ile Leu 45 50 55 GAT GGA ATA GAC TGC ACA CTG ATA GAT GCT CTA TTG GGG GAC CCT 307 Asp Gly Ile Asp C ys Thr Leu Ile Asp Ala Leu Leu Gly Asp Pro 60 65 70 CAT TGT GAT GTT TTT CAA AAT GAG ACA TGG GAC CTT TTC GTT GAA 352 His Cys Asp Val Phe Gln Asn Glu Thr Trp Asp Leu Phe Val Glu 75 80 85 CGC AGC AAA GCT TTC AGC AAC TGT TAC CCT TAT GAT GTG CCA GAT 397 Arg Ser Lys Ala Phe Ser Asn Cys Tyr Pro Tyr Asp Val Pro Asp 90 95 100 TAT GCC TCC CTT AGG TCA CTA GTT GCC TCG TCA GGC ACT CTG GAG 442 Tyr Ala Ser Leu Arg Ser Leu Val Ala Ser Ser Gly Thr Leu Glu 105 110 115 TTT ATC ACT GAG GGT TTC ACT TGG ACT GGG GTC ACT CAG AAT GGG 487 Phe Ile Thr Glu Gly Ghe Phe Thr Trp Thr Gly Val Thr Gln Asn Gly 120 125 130 GGA AGC AAT GCT TGC AAA AGG GGA CCT GGT AGC GGT TTT TTC AGT 532 Gly Ser Asn Ala Cys Lys Arg Gly Pro Gly Ser Gly Phe Phe Ser 135 140 145 AGA CTG AAC TGG TTG ACC AAA TCA GGA AGC ACA TAT CCA GTG CTG 577 Arg Leu Asn Trp Leu Thr Lys Ser Gly Ser Thr Tyr Pro Val Leu 150 155 160 AAC GTG ACT ATG CCA AAC AAT GAC AAT TTT GAC AAA CTA TAC ATT 622 Asn Val Thr Met Pro Asn Asn Asp Asn Phe Asp Lys Leu Tyr Ile 165 170 175 T GG GGG ATT CAC CAC CCG AGC ACG AAC CAA GAA CAA ACC AGC CTG 667 Trp Gly Ile His His Pro Ser Thr Asn Gln Glu Gln Thr Ser Leu 180 185 190 TAT GTT CAA GCA TCA GGG AGA GTC ACA GTC TCT ACC AGG AGA AGC 712 Tyr Val Gln Ala Ser Gly Arg Val Thr Val Ser Thr Arg Arg Ser 195 200 205 CAG CAA ACT ATA ATC CCG AAT ATC GGG TCC AGA CCC TGG GTA AGG 757 Gln Gln Thr Ile Ile Pro Asn Ile Gly Ser Arg Pro Trp Val Arg 210 215 220 GGT CTG TCT AGT AGA ATA AGC ATC TAT TGG ACA ATA GTT AAG CCG 802 Gly Leu Ser Ser Arg Ile Ser Ile Tyr Trp Thr Ile Val Lys Pro 225 230 235 GGA GAC GTA CTG GTA ATT AAT AGT AAT GGG AAC CTA ATC GCT CCT 847 Gly Asp Val Leu Val Ile Asn Ser Asn Gly Asn Leu Ile Ala Pro 240 245 250 CGG GGT TAT TTC AAA ATG CGC ACT GGG AAA AGC TCA ATA ATG AGG 892 Arg Gly Tyr Phe Lys Met Arg Thr Gly Lys Ser Ser Ile Met Arg 255 260 265 TCA GAT GCA CCT ATT GAT ACC TGT ATT TCT GAA TGC ATC ACT CCA 937 Ser Asp Ala Pro Ile Asp Thr Cys Ile Ser Glu Cys Ile Thr Pro 270 275 280 280 AAT GGA AGC ATT CCC AAT GAC AAG CCC TTT CAA AACGTA AAC AAG 982 Asn Gly Ser Ile Pro Asn Asp Lys Pro Phe Gln Asn Val Asn Lys 285 290 295 ATC ACA TAT GGA GCA TGC CCC AAG TAT GTT AAG CAA AAC ACC CTG 1027 Ile Thr Tyr Gly Ala Cys Pro Lys Tyr Val Lys Gln Asn Thr Leu 300 305 310 AAG TTG GCA ACA GGG ATG CGG AAT GTA CCA GAG AAA CAA ACT AGA 1072 Lys Leu Ala Thr Gly Met Arg Asn Val Pro Glu Lys Gln Thr Arg 315 320 325 GGC CTA TTC GGC GCA ATA GCA GGT TTC ATA GAA AAT GGT TGG GAG 1117 Gly Leu Phe Gly Ala Ile Ala Gly Phe Ile Glu Asn Gly Trp Glu 330 335 340 GGA ATG ATA GAC GGT TGG TAC GGT TTC AGG CAT CAA AAT TCT GAG 1162 Gly Met Ile Asp Gly Trp Tyr Gly Phe His Gln Asn Ser Glu 345 350 355 GGC ACA GGA CAA GCA GCA GAT CTT AAA AGC ACT CAA GCA GCC ATC 1207 Gly Thr Gly Gln Ala Ala Asp Leu Lys Ser Thr Gln Ala Ala Ile 360 365 370 GAC CAA ATC AAT GGG AAA TTG AAC AGG GTA ATC GAG AAG ACG AAC 1252 Asp Gln Ile Asn Gly Lys Leu Asn Arg Val Ile Glu Lys Thr Asn 375 380 385 GAG AAA TTC CAT CAA ATC GAA AAG GAA TTC TCA GAA GTA GAA GGG 1297 Glu Lys Phe His Gln Ile G lu Lys Glu Phe Ser Glu Val Glu Gly 390 395 400 AGA ATT CAG GAC CTC GAG AAA TAC GTT GAA GAC ACT AAA ATA GAT 1342 Arg Ile Gln Asp Leu Glu Lys Tyr Val Glu Asp Thr Lys Ile Asp 405 410 415 CTC TGG TCT TAC AAT GCG GAG CTT CTT GTC GCT CTG GAG AAT CAA 1387 Leu Trp Ser Tyr Asn Ala Glu Leu Leu Val Ala Leu Glu Asn Gln 420 425 430 CAT ACA ATT GAC CTG ACT GAC TCG GAA ATG AAC AAG CTG TTT GAA 1432 His Thr Ile Asp Leu Thr Asp Ser Glu Met Asn Lys Leu Phe Glu 435 440 445 AAA ACA AGG AGG CAA CTG AGG GAA AAT GCT GAA GAG ATG GGC AAT 1477 Lys Thr Arg Arg Gln Leu Arg Glu Asn Ala Glu Glu Met Gly Asn 450 455 460 GGT TGC TTC AAA ATA TAC CAC AAA TGT GAC AAC GCT TGC ATA GAG 1522 Gly Cys Phe Lys Ile Tyr His Lys Cys Asp Asn Ala Cys Ile Glu 465 470 475 TCA ATC AGA AAT GGT ACT TAT GAC CAT GAT GTA TAC AGA GAC GAA 1567 Ser Ile Arg Asn Gly Thr Tyr Asp His Asp Val Tyr Arg Asp Glu 480 485 490 GCA TTA AAC AAC CGG TTT CAG ATC AAA GGT GTT GAA CTG AAG TCT 1612 Ala Leu Asn Asn Arg Phe Gln Ile Lys Gly Val Glu Leu Lys Ser 495 5 00 505 GGA TAC AAA GAC TGG ATC CTG TGG ATT TCC TTT GCC ATA TCA TGC 1657 Gly Tyr Lys Asp Trp Ile Leu Trp Ile Ser Phe Ala Ile Ser Cys 510 515 520 TTT TTG CTT TGT GTT GTT TTG CTG GGG TTC ATC ATG TGG GCC TGC 1702 Phe Leu Leu Cys Val Val Leu Leu Gly Phe Ile Met Trp Ala Cys 525 530 535 CAG AGA GGC AAC ATT AGG TGC AAC ATT TGC ATT TGAGTGTATT AGTAATTAAA 1755 Gln Arg Gly Asn Ile Arg Cys Asn Ile Cys IleCT TAG TGCT 545 CCG 1778

SEQ ID NO: 55 Sequence length: 20 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid (synthetic DNA) Sequence ATTGTTGCAT ATTTTCCCCG 20

SEQ ID NO: 56 Sequence length: 20 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid (synthetic DNA) Sequence ATTGATACCT GTATTTCTGA 20

SEQ ID NO: 57 Sequence length: 1110 Sequence type: Nucleic acid Number of strands: Double-stranded Topology: Linear Sequence type: cDNA to genomic RNA Origin: Organism name: A2 / Aichi / 2/68 SEQ CTAGAAGCAA AGCAGGGGAT AATTCTATTA ATCATGAAGA CCATCATTGC TTTGAGCTAC 60 ATTTTCTGTC TGGCTCTCGG CCAAGACCTT CCAGGAAATG ACAACAGCAC AGCAACGCTG 120 TGCCTGGGAC ATCATGCGGT GCCAAACGGA ACACTAGTGA AAACAATCAC AGATGATCAG 180 ATTGAAGTGA CTAATGCTAC TGAGCTAGTT CAGAGCTCCT CAACGGGGAA AATATGCAAC 240 AATATTGATA CCTGTATTTC TGAATGCATC ACTCCAAATG GAAGCATTCC CAATGACAAG 300 CCCTTTCAAA ACGTAAACAA GATCACATAT GGAGCATGCC CCAAGTATGT TAAGCAAAAC 360 ACCCTGAAGT TGGCAACAGG GATGCGGAAT GTACCAGAGA AACAAACTAG AGGCCTATTC 420 GGCGCAATAG CAGGTTTCAT AGAAAATGGT TGGGAGGGAA TGATAGACGG TTGGTACGGT 480 TTCAGGCATC AAAATTCTGA GGGCACAGGA CAAGCAGCAG ATCTTAAAAG CACTCAAGCA 540 GCCATCGACC AAATCAATGG GAAATTGAAC AGGGTAATCG AGAAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGA GAGA GA TCTCTGGTCT TACAATGCGG AGCTTCTTGT CGCTCTGGAG 720 AATCAACATA CAATTGACCT GACTGACTCG GAAATGAACA AGCTGTTTGA AAAAACAAGG 780 AGGCAACTGA GGGAAAATGC TGAAGAGATG GGCAATGGTT GCTTCAAAAT ATACCACAAA 840 TGTGACAACG CTTGCATAGA GTCAATCAGA AATGGTACTT ATGACCATGA TGTATACAGA 900 GACGAAGCAT TAAACAACCG GTTTCAGATC AAAGGTGTTG AACTGAAGTC TGGATACAAA 960 GACTGGATCC TGTGGATTTC CTTTGCCATA TCATGCTTTT TGCTTTGTGT TGTTTTGCTG 1020 GGGTTCATCA TGTGGGCCTG CCAGAGAGGC AACATTAGGT GCAACATTTG CATTTGAGTG 1080 TATTAGTAAT TAAAAACACC CTTGTTTCTG 1110

SEQ ID NO: 58 Sequence length: 346 Sequence type: amino acid Number of chains: single-chain Topology: linear Sequence type: peptide Sequence Met Lys Thr Ile Ile Ala Leu Ser Tyr Ile Phe Cys Leu Ala Leu -15 -10 -5 Gly Gln Asp Leu Pro Gly Asn Asp Asn Ser Thr Ala Thr Leu Cys 1 5 10 Leu Gly His His Ala Val Pro Asn Gly Thr Leu Val Lys Thr Ile 15 20 25 Thr Asp Asp Gln Ile Glu Val Thr Asn Ala Thr Glu Leu Val Gln 30 35 40 Ser Ser Ser Thr Gly Lys Ile Cys Asn Asn Ile Asp Thr Cys Ile 45 50 55 Ser Glu Cys Ile Thr Pro Asn Gly Ser Ile Pro Asn Asp Lys Pro 60 65 70 Phe Gln Asn Val Asn Lys Ile Thr Tyr Gly Ala Cys Pro Lys Tyr 75 80 85 Val Lys Gln Asn Thr Leu Lys Leu Ala Thr Gly Met Arg Asn Val 90 95 100 Pro Glu Lys Gln Thr Arg Gly Leu Phe Gly Ala Ile Ala Gly Phe 105 110 115 Ile Glu Asn Gly Trp Glu Gly Met Ile Asp Gly Trp Tyr Gly Phe 120 125 130 Arg His Gln Asn Ser Glu Gly Thr Gly Gln Ala Ala Asp Leu Lys 135 140 145 Ser Thr Gln Ala Ala Ile Asp Gln Ile Asn Gly Lys Leu Asn Arg 150 155 160 Val Ile Glu Lys Thr Asn Glu Lys Phe His Gln Ile Glu Lys Glu 165 170 175 Phe Ser Glu Val Glu Gly Arg Ile Gln Asp Leu Glu Lys Tyr Val 180 185 190 Glu Asp Thr Lys Ile Asp Leu Trp Ser Tyr Asn Ala Glu Leu Leu 195 200 205 Val Ala Leu Glu Asn Gln His Thr Ile Asp Leu Thr Asp Ser Glu 210 215 220 Met Asn Lys Leu Phe Glu Lys Thr Arg Arg Gln Leu Arg Glu Asn 225 230 235 Ala Glu Glu Met Gly Asn Gly Cys Phe Lys Ile Tyr His Lys Cys 240 245 250 Asp Asn Ala Cys Ile Glu Ser Ile Arg Asn Gly Thr Tyr Asp His 255 260 265 Asp Val Tyr Arg Asp Glu Ala Leu Asn Asn Arg Phe Gln Ile Lys 270 275 280 Gly Val Glu Leu Lys Ser Gly Tyr Lys Asp Trp Ile Leu Trp Ile 285 290 295 295 Ser Phe Ala Ile Ser Cys Phe Leu Leu Cys Val Val Leu Leu Gly 300 305 310 Phe Ile Met Trp Ala Cys Gln Arg Gly Asn Ile Arg Cys Asn Ile 315 320 325 Cys Ile 330

[Brief description of the drawings]

FIG. 1 is a schematic diagram of the tertiary structure of an HA molecule and a diagram showing the position of a common conserved region in the HA molecule between H1N1 and H2N2 subtypes.

FIG. 2 is a schematic diagram of the tertiary structure of the HA molecule and a diagram showing the position of a common conserved region in the HA molecule of the H3N2 subtype.

FIG. 3 is a view showing the survival rate of an influenza virus infection group.

FIG. 4 is a view showing the survival rate of an influenza virus infection group.

FIG. 5 is a graph showing changes in body weight of an influenza virus infection group.

FIG. 6 is a view showing the survival rate of a group infected with influenza virus.

Continuation of the front page (51) Int.Cl. ⁷ Identification symbol FI // C12P 21/08 C12P 21/08 (56) References JP-A-6-100594 (JP, A) Ann. Rev .. Biochem. , 56 (1987), p. 365-394J. Gen. Virol. , 68 (1987), (8), p. 2249-2252 Nature, 287 (1980), (5780), p301-306 Nature, 292 (1981), (5818), p72-75 (58) Fields investigated (Int. Cl. ⁷ , DB name) C12N 15 / 00 BIOSIS (DIALOG) CA (STN) REGISTRY (STN) WPI (DIALOG)

Claims (5)

(57) [Claims] 1. An immunogenic artificial polypeptide having substantially the same antigenicity as a stem region in a hemagglutinin molecule of a human influenza A virus and having a deletion of a globular region in the hemagglutinin molecule. 2. SEQ ID NO: 1 in the sequence listing at least in the molecule.
And a GITNKVNSVIEK polypeptide sequence represented by SEQ ID NO: 2 in the sequence listing, and the configuration of both sequences is H1N1 subtype and H2
The artificial immunogenic polypeptide according to claim 1, wherein the polypeptide has substantially the same antigenicity as the stem region in the hemagglutinin molecule of the N2 subtype. 3. The compound having at least SEQ ID NO.
And a QINGKLNR (L / V) IEK polypeptide sequence represented by SEQ ID NO: 4 in the sequence listing, wherein the configuration of both sequences is a stem in a hemagglutinin molecule of the H3N2 subtype. 2. The immunogenic artificial polypeptide according to claim 1, which has substantially the same antigenicity as the region. 4. The artificial immunogenic polypeptide according to claim 1, which is separated from a protease-treated product of a human influenza A virus hemagglutinin molecule. 5. A gene encoding an immunogenic artificial polypeptide having substantially the same antigenicity as a stem region in a hemagglutinin molecule of a human influenza A virus and having a deletion of a globular region in the hemagglutinin molecule.