JPH01193228A - Recombinant htl v-iii protein and its use - Google Patents

Abstract

PURPOSE: To provide a recombinant HTLV-III envelope protein useful for the diagnosis, prevention and treatment of AIDS. CONSTITUTION: Plasmid pBR10 containing 1,275 base pairs from the Kpnl position to the BglII position of a DNA coding HTLV-III env gene, plasmid pPBl containing 540 base pairs from the PvuII position to the Bglll position, plasmid p590 containing 1,055 base pairs from the PvuII position to the HindIII position and plasmid pKH1 containing 1, 830 base pairs from the KpnI position to the HindIII position of the above DNA are expressed by using a proper host such as E.coli to obtain new recombinant HTLV-III fused protein RIO (95KD), PBI. (26KD), 590 (86KD) and KHl(70KD). The protein stimulates the lymphocyte proliferation response of man infected with HTLV-III to stimulate the immune system and respond to HTLV-III.

Description

[Detailed description of the invention] [Industrial application field] The present invention relates to recombinant HTLV-m protein and its uses.

[Prior Art] Human T-cell leukemia virus (HTLV-m), lymphadenopathy-associated virus (LAV), or AIDS-associated retrovirus (ARV) has been linked to acquired immunodeficiency disease (AIDS).
) [BobobicφM (Pop
ovic + gate), Sarungadharan M Kenji (S
arngadha-ran, M.G.), Read, E., and Garo R.C.
allo, R, C,) [1984] 5cienc
e224, pp. 497-500], this virus exhibits a tropism for the T4'' lymphocyte subset [Clararaman, D., Barr.
Barre-5inoussi, F
,), NBeyre MΦT (NBeyre, M,
T,), Dauguet.

C,), VilIIer, E,
, Griscelli, C., Brun-Vezinet: r-F.
, F,), Rouzioux*C,)
, Gluckman,
J.

C, ), Cx) Leman G.C.
n, J, C,) and Montagnière-2 Les (Mont.
agnier, L-) (1984) 5science
225, pp. 59-63], most AIDS patients, AIDS-related complex syndrome (ABC) patients, and asymptomatic people infected with the virus [Sarungadharan M.
G, Bobobic M, Pratch L (8ruch
, L.), Sch-upba J.
ch, J) and Gallo, R.C.
R,C,) (1984) Science 224 5
G+3-508] antigens against the pore v-■ proteins in serum have enabled the development of immunologically based tests to detect antibodies against these antigens. These tests are used to reduce the transmission of HTLV-m through blood transfusions by examining blood samples from people infected with the virus. Currently commercially available diagnostic tests use inactivated viral proteins as antigens.

In addition to providing new clues to diagnosis, recombinant A-A holds great promise for the development of vaccines against both bacteria and viruses [Wilson T.
on, T.) [1984] Bio/Techno
2, pp. 29-39] The most widely used organism for expressing recombinant vaccines is Escherichia coli (E, c
oli), S.

cerevishi x (s, cerevtstae) and culture!
III Mammalian cells such as 1, limb 0 disease [Creit D, G, Yansura+D, Small, 8.] Daubenko ψ de 4- (
Dowbenko, D.), Moore Φ de 4--x M (
Moore, D. M.), Brapman M.G.
Brub-man, M, J,), Mc-niercher, P, D,), Mor-gan*D, 0-
Robert-son,
B, 11. ), and Packlatch H.L. (B
c11racl+, H, L, ) (1981) Scie
nce 214, pp. 1125-1129)] and malaria [Young.

, I, F, ), Hoekmeyer, V, T., Gross, M., Ripley Ba1lou, W. (Wirtz, R.A.), Trosperp J.
・Beaudoin, R
, L.), Hollin M.R.
Dale, M, R,), Miller LM (formerly 1l
er, L, M-), Diggs
gs, C, L, ), and 6-Senberg M (Ro
senber3. M.) (1985) 5cien
ce, Vol. 228, pp. 958-962) was synthesized in Escherichia coli. Other examples are hepatitis B surface antigens produced in yeast [McAleere V, J., Buynak, E.;

B, ), Maigetter e R Φ set (Maiget
ter, R.

Z, ), '7 Sampler DA (WaBIer)
, D, E, ), Mirror Double
ere IJ-”,) and Hillman Life M.R.
lil'leman, Fl, R, 81984) N
ature 30? Volume 178-180], and herpes vaccines made with mammalian cells [Berman B.
Robert, P., I. J., Gregory T., Chase, D., and Lasky, L. A. (1984). ) Science
ce, Vol. 227, Otori, pp. 4O0-1492].

[Problem B to be solved by the invention] As of 2001, there is an urgent need to develop an AIDS vaccine. No such vaccine is known to exist.

[Means for solving the problem] The present invention provides a novel recombinant HTLV-IH protein and its IH protein.
Regarding J use. More specifically, the present invention relates to diagnosis of AIDS,
Novel recombinant HTLV-II that can be used for prevention or treatment
I between envelope proteins. Furthermore, the recombinant IITLV-III envelope protein of the present invention
It can then be used to stimulate the lymphocyte proliferative response in humans infected with A-111, which then stimulates the immune system to respond to IITLV-m in these humans, and thus the enveloping A-butanbac fragment is protective. You can call 1M and get 1a.
It becomes something with l+I III. These novel proteins can be coated into bacterial plasmids using standard procedures and used to transform a suitable host, such as E. coli.

Expression vector plasmid pREV2.2 was transformed into plasmid p
Constructed from BG+. A flow diagram illustrating the construction of this plasmid is shown in Figure 1 of the drawings.

Plasmid pR1o consists essentially of Bgl from the pn1 position.
encoded the HTLV-m env genes up to II [
Contains 1NAPJ+275 base pairs. This plasmid in a suitable bacterial host, e.g.
A novel recombinant IITLV of 95 kD designated as 10
-m can be used to create fusion proteins. The amino acid sequence of the fusion protein RIO is shown in Table 8. The DNA sequence encoding this protein is shown in Table 8A.
The amino acid sequence of the 111v portion of 10 is shown in Table 12.
The FIN^ sequence encoding the 1V portion of protein R10 is shown in Table 12A.

Plasmid pPBI contains approximately 540 base pairs of DNA encoding the HTLV-m env gene, essentially from the Pvu11 position to the Bgl■ position, and is designated FBI using this plasmid in a suitable host, such as E. coli. A novel silk recombinant IITLV-111 fusion protein of 26 kD can be created. Fusion protein Pal
The amino acid sequence of protein Pa is shown in Table 9. The DNA sequence encoding this protein is shown in Table 9A. The amino acid sequence of the old V portion of protein PBI is shown in Table 13.
The DNA sequence encoding the HIV portion of 1 is shown in Table 13A.

Plasmid 11590 essentially extends from the Pvu■ position to the Hi
ndIII II HTLV-III env up to 2 positions
It contains approximately 1055 base pairs of DNA encoding the gene. Using this plasmid in a suitable host, such as E. coli, a novel recombinant IITLV-m protein of 811 ko, designated 590, can be made. The amino acid sequence of fusion protein 590 is shown in Table 1n. The DNA sequence encoding this protein is shown in Table 10A. The amino acid sequence of the l-11V portion of protein 590 is shown in Table 14.
The DNA sequence that coats the 111v portion of protein 590 shown in the table is shown in Table 14A.

Plasmid I] ■1 is essentially 1lin from the Kpn 1 position old II k? IITLV-[11en
vil fF, which encoded the child [lNA contains approximately 1830 base pairs. Using this plasmid in a suitable host, such as ζJ E. coli, a novel recombinant IITLV-m protein of TOkD, designated Kold, can be generated. ■For fusion protein! The amino acid sequence of this protein is shown in Table 11.
Shown below. The amino acid sequence of the HIV portion of Hl in the protein is shown in Table 15, and the DNA sequence encoding the old ■1v portion of the protein is shown in Table 15A.

The Bushmid peGt in the large IIIW host MS371 is deposited at the US Department of Agriculture Northern Regional Research Laboratory (NRRL), Biolia, IL, USA. The plasmid is in the permanent storage facility of this depository. E. coli MS371 (pBG
l).

NRRL B-15904 is the month of 1984! It was deposited on the day. Daibiki IMS3?1. NRRL B-151
29 is currently a type of bacterium III 5G20 that can generally be done manually.
251. NRRL B-15918, 1984
It was deposited on February 12th. NRRL B-15904
and NRRL B-15918 will be available to the public through the granting of patents disclosing them.Other culture media deposited with NRRL and their deposit dates and numbers are as follows:

The above deposits will be kept in the NRRL Trust Facility for at least 30 years and will be available to the public upon the granting of patents disclosing them; Can be done manually as required by patent law. However, it should be understood that the availability of the deposit does not constitute a license to practice the invention in violation of patent rights granted by government action.

The novel II T LV m protein of the present invention is derived from Saccharomyces Φ cerevisiae.
A plasmid containing an inducible galactose promoter from A. cerevisiae can be used to effect expression in this microorganism [Broach, 1.R, Lee et al.
Wai (Li, Y,), Wu L C (vu, L)
, C,), and Jayaram M (''ayara+m,
M.) Experimental Manipulation of r Gene Expression J (1983) p. 83, edited by M. Inouni, Academic Press].

These plasmids are called YEp51 and VEp52 [Broch J.R. et al. (1983)];
Big ++ ii compound I! starting point, β-lactamase gene,
Yeast La12 gene, 2μm duplication! 1 starting point, and 2μ−
Contains circular REP3 positions. Expression of the &[I transgene is driven by the yeast GALIO gene promoter.

The yeast promoter is a promoter of galactose and alcohol dehydrogenase [Benne
Tzen, J.L.) and Hal B.D. (H.
all, B. Mouth, ) (1982) J. Biol.
Chew, vol. 257, p. 3018;
Amerer, G.)r Enzymology Methods” (Meth
ods in EnzymologyX1983)
101, p. 192], phosphoglycerate competition kinase [
Deryck, R, Hitzes+an, R,A
), Gray, P, V
), Goed-del Fist 4- (Goed-del
, D.V.), Experimental Manipulation of Gene Expression J (1983
), p. 247, edited by M Inouuni, Academic Press], triose phosphate isomerase [
Alber, T., and Kawasaki, G. (1982) J., Molec, and A.
pplied Genet, Vol. 1, p. 419], or enolase [Innes, M.A.
(1985) Science Vol. 226, p. 21] can be used for one history.

The genes revealed herein can generate IQ in simian cells. The genes encoding these proteins were reported by Okayama and Barb [Okayama, H.

and Berg, P. (1983) Molec,
and Ce11.101.3, page 280] and Part 4
Synthesis of HTLV-III protein can be detected immunologically when cloned into CIJS cells transformed with one of the plasmids described in the references or these plasmids.

Other mammalian cell gene expression protein production systems can be used. fli! Examples of such systems include the vaccinia virus expression system [Moss, B.
1 spirit)) 15 years) I*munoloxy Today
Volume 6, p. 243; Chakraparti Nis (CI+akr)
abarti, S.), Pretschling Kay (B.
reclling, ), and Mosby (19
1985) Molec, and theft, Bio
1, Vol. 5, p. 3403] and vectors derived from murine retroviruses [Mulligan R.C.
llingan, R. (,) Experimental Manipulation of Gene Valve IQ J (1983), p. 155, M. Inouuni, Academia Press, Inc.].

The HTLV・■ protein of the present invention is BOC and FMOC [Merrifield, R.B.
R,B,) (1963) J, Ataer, Ch.
ew, Soc, vol. 85, p. 2149; Zhang Xi (
Chang, C.) and Meienhofer, J. (1978) In
t, J, Peptide Protein Res
, Vol. 11, p. 246].

As is well known in the art, the amino acid sequence of a protein is
[Determined by the nucleotide sequence of lNA. Because of the redundancy of the genetic code, that is, more than one coded nucleotide triplet (codon) can be used for most of the amino acids used to make proteins, different nucleotide sequences can be used for one particular amino acid. can be coded. Therefore, the genetic code can be depicted as follows.

Phenylalanine (Pbe) TT and leucine (L
eu) XTY isoleucine (lle)
ATM methionine (MeL) A
TG valine (Vat) GTL serine (Ser) GRS proline (1'
r o ) CCI.

Threonine (TI+r) ACL Alanine (
Ala) GCL Tyrosine (Tyr)
Histidine (llis) in TA
Glutamine (Gln) in CA CAJ asparagine (^sn) Lysine (Lys) in AA
AAJ Aspartic Acid (Asp)
GAK glutamic acid (GILI) CA
J cysteine (Cys) TG tryptophan (Trp) TGG arginine (Arg)
VGZ glycine (Gly)
+; GL termination signal TAJ termination signal TGA Interpretation method: Each three-letter deoxynucleotide triplet is
Corresponds to the trinucleotide of the messenger RNA with the 59 end on the left and the 3' end on the right. All DNAs described herein are derived from DNA strands whose sequence corresponds to that of mRNA, with uracil replaced by thymine. The zero character indicates a purine or pyrimidine base forming a deoxynucleotide sequence.

A = adenine 6=guanine C: Cytosine T=thymine If Y is A or 6, then X=T or 1:.

If V is C or T, then X=C.

If X is C, then Y=A, G, C or T.

If X is T, Y=A or 6° If Z is A or G, V=(: or A6Z is C or T
In the case of , V=C.

If the enemy is C, Z=A, G, C or T.

If Suga is A, then Z=A or G.

If S is A, G, C or T then QR=TC, or alternatively if S is T or C then QRFAG.

, 1=A shank ni=T or C 1, = A, T, C or G.

M=A, l: or T.

] 2 shows that the novel amino 1 Iltl sequence of the HTLV-[11 protein of the present invention can be prepared by a nucleotide sequence other than that revealed by Kikiri**. These IITLV-m proteins, or HTLV-
A novel amino acid sequence of anti-JH: ii9 or a fragment thereof with immunogenic or therapeutic activity.・Functionally identical nucleotide sequences can be produced using known synthetic procedures. The present invention encompasses such functionally equivalent nucleotide sequences.

Thus, the scope of the present invention extends not only to the specific nucleotide sequences described in Kikiri *sho, but also to substantially the same
Also included are all equivalent nucleotide sequences that encode molecules with TLV-Ill antigenic or immunogenic or therapeutic activity.

Furthermore, the scope of the present invention not only includes the specific amino acid sequences identified, but also includes proteins that have a significant ability to induce the formation of and/or bind to specific IITLV-1 antibodies. or similar sequences encoding protein fragments.

The term "equivalent," as used in common patent terminology, refers to molecules that have substantially the same HTLV-m antigenic or immunogenic or therapeutic activity in essentially the same type of host. is used to refer to a nucleotide sequence that substantially serves as the nucleotide sequence identified herein to produce. Also included within the scope of this definition are secondary fragments of IITLV-111 that have antigenic or immunogenic or therapeutic activity.

As revealed above, by microbial methods I
In order to make ITLV-III protein, the H
The use of nucleotide sequences encoding antigenic or immunogenic or therapeutic activity of TLV-m is well known to those skilled in the art of genetic engineering by fusing the sequences into expression vectors in eukaryotes (yeast or mammalian cells). ) or prokaryotes (Mato Wa
Transformation or transfection of I cells into a host is performed using other well-known proteins such as insulin, interferon, human growth hormone, I-1, IL-2, etc. It is standard procedure. Similar procedures, or obvious variations thereof, can be used in accordance with the present invention to produce HTLV-m proteins by microbial means or tissue culture techniques.

The nucleotide sequences disclosed herein can be produced by "genetic machinery" by procedures well known in the art. This is possible because of the disclosure of the nucleotide sequence.

The disclosed restriction enzymes can be purchased from Bethesda Laboratories (Gaithersburg, MD) or New England Φ Biolabs (Beverly, MA).

Enzymes are used according to the instructions provided by the supplier.

The various methods used to prepare plasmids and transform host organisms are well known in the art. All these steps were performed by Maniatl.
s+T, ), Fri-tsch
, E, F.) and Samprutsk, J. (Sasbr.
ook.

(1982) "Molecular Cloning" (Experimental Manual) Cold Spring Harbor Laboratory, New York). In this way, DNA is extracted from 0NAlt microbial cells and digested with restriction enzymes.
The fragments are subjected to electrophoresis, the plasmid is tailed and annealed, the DNA is inserted, the DNA is recombined, cells are transformed, e.g. large am cells, and plasmid D
It is within the skill of those skilled in the art to prepare the NA, subject the proteins to electrophoresis, and sequence the DNA.

Immunochemical assays using the HTLV-III proteins of the invention can take a variety of forms. A preferred type is a solid phase immunoassay. For this type of test, the HTLV
-m protein is immobilized on a solid phase to form an antigen-immunoadsorbent. The immunoadsorbent is incubated with the sample to be tested. After an appropriate incubation period, the immunoadsorbent is separated from the sample and a labeled anti-(human) IgG) antibody is used to detect human anti-HTLV-III bound to the immunoadsorbent.
Detect antibodies. Comparing the amount of label associated with the immunoadsorbent to positive and negative controls allows an assessment of the presence or absence of anti-HTLV-III antibodies.

Immunoadsorbents are made by adsorbing or binding purified HTLV-m protein to a solid phase.

A variety of solid phases can be used, such as beads of glass, polystyrene, polypropylene, dextran, or other materials. Other suitable solid phases include tubes or plates made of or coated with these materials.

HTLV-m proteins can be covalently or non-covalently attached to solid phases by techniques such as covalent bonding via amide or ester linkages or by adsorption. After the HTLV-III protein has been immobilized on the solid phase, the solid phase can be post-coated with an animal protein, such as 3χ fish gelatin. This provides a sequestering protein that reduces non-specific adsorption of proteins to the immunoadsorbent surface.

The immunoadsorbent is then incubated with the sample to be tested for anti-HTLV-III antibodies. Blood tests use plasma or serum. Dilute the plasma or serum with normal animal plasma or serum. The diluted plasma or serum is derived from the same animal species that is the source of the anti-(human IgG) antibody. A preferred anti-(human 18G) antibody is a goat anti-(human IgG) antibody. Thus, in a preferred embodiment, the diluent is goat serum or plasma.

The culture conditions, such as pH and temperature, as well as the culture period, are not critical; these parameters can be optimized by routine experimentation. Generally, culturing is carried out in a pH 7-8 buffer at about 45°C for 1-2 hours.

After culturing, the immunoadsorbent and sample are separated. Separation can be performed by conventional separation techniques such as sedimentation or centrifugation, and the immunoadsorbent can be washed free of sample to eliminate interfering substances.

To detect human antibodies bound to an immunoadsorbent, the immunoadsorbent is combined with a labeled anti-(human) IgG) antibody (tracer).
Culture together. Generally, the immunoadsorbent is incubated with a labeled anti-(human) IgG) antibody solution containing a small amount (approximately 1x) of serum or plasma of the animal species that serves as the source of the anti-(human) IgG) antibodies. Anti-(human) IgG) antibodies can be obtained from any animal source. However, goat anti-(human 18G) antibodies are preferred. Anti-(human IgG) antibodies are human) IgG F
Antibodies against the c fragment, e.g. goat anti-(human IgG)F
c antibody.

The anti-(human) IgG) antibody or anti-(human) IgG) Fc can be labeled with a radioactive material, such as iodine 19125; with an optical label such as a fluorescent material; or with an enzyme such as horseradish peroxidase. . Anti-human antibodies can also be biotinylated and their binding to immunoadsorbents detected using labeled avidin.

After incubation with the labeled antibody, the immunoadsorbent is separated from the Na solution and the label associated with the immunoadsorbent is evaluated. Depending on the choice of label, evaluation can be performed in a variety of ways. If the label is a radioactive gamma emitter, it can be detected with a gamma counter, or if it is a fluorescent material, it can be detected with a fluorometer. In the case of enzymes, label detection is performed using enzyme substrates.
This can be done by colorimetric method.

To determine the presence of anti-HTLV-m antibodies, the amount of label bound to the immunoadsorbent is compared to positive and negative controls. A control group is generally treated at the same time as the test sample. The positive control is serum containing anti-HTLV-m antibodies. Negative controls are sera from healthy people that do not contain anti-HTLV-III antibodies.

For convenience and standardization, reagents for performing immunoassays can be combined into assay kits. Blood test kits include, for example, the following:

(a) an immunoadsorbent, e.g. polystyrene beads coated with HTLV m protein; (b) a diluent for serum or plasma samples, e.g. normal serum or plasma; (c) anti-(human IgG) antibodies; For example, goat anti (human IgG) in a buffered aqueous solution containing about IK of goat serum or plasma.
) antibodies; (d) positive controls, e.g., serum containing antibodies to at least one of the novel HTLV-m proteins; and (e) negative controls, e.g., no antibodies to at least one of the novel HTLV-m proteins. Stored serum from healthy individuals.

If the label is an enzyme, the additional component of the kit can be a substrate for the enzyme.

Another type of anti-HTLV-III antibody assay is the antigen sandwich assay. This assay uses anti-(human Ig
G) Anti-HTLV-II conjugated to immunoadsorbent using labeled HTLV-III protein instead of antibody
Detect I antibodies. This assay is essentially based on the bivalency of the antibody molecule. One binding site of the antibody associates with the antigen immobilized on the solid phase. The second position is available for binding labeled antigen.

The assay procedure is essentially the same as that described for the immunoassay, except that after incubation with the sample, the immunoadsorbent is
Incubate with TLV-m protein solution. For this type of assay, the HTLV-m protein can be labeled with radioisotopes, enzymes, etc.

In a third format, I
The bacterial protein protein A, which binds to the Fc fragment of the gG molecule, can be used as a labeled tracer to detect anti-HTLV-III antibodies adsorbed to the immunoadsorbent. Protein A can be easily labeled with radioisotopes, enzymes or other detectable species.

Immunochemical assays using HTLV-m protein have several advantages over those using whole (or crushed) virus. An assay based on the HTLV-m protein would alleviate concerns about the large amount of infectious virus being grown and the inherent diversity associated with cyst culture and virus production; This will help alleviate the real or perceived fear of contracting AIDS among skilled workers.

Vaccines comprising one or more of the HTLV-m proteins disclosed herein, and antigenic variants thereof, can be made by procedures well known in the art, e.g. can be prepared as injectable solutions, such as liquid solutions or suspensions. Solid forms can also be prepared for solution in, or suspension in, liquid prior to injection. Optionally, the formulation can also be emulsified. The active antigenic component can be mixed with pharmaceutically acceptable adjuvants which are compatible with the active ingredient. Examples of suitable excipients are water, saline, dextrose, glycerol, ethanol, etc., and combinations thereof. Furthermore, if desired, the vaccine can contain small amounts of auxiliary substances such as wetting agents, emulsifying agents, pH buffering agents, and auxiliary agents that enhance the effectiveness of the vaccine. It is convenient to administer the vaccine parenterally by injection, eg subcutaneously or intramuscularly. Additional prescription drugs suitable for Ike's mode of administration are suppositories,
In some cases, it is also an oral prescription drug. Traditional binders and carriers for suppositories include, for example, polyalkylene glycols or triglycerides. Suppositories can be formed from mixtures containing active ingredients in the range of about 0.5z to about 10z1, preferably about 1x to about 2z. Oral formulations can include commonly used adjuvants such as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, and the like. These compositions may take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations, or powders and contain from about 10% to about 95%, preferably from about 25% to about 70% active ingredient. .

Proteins can be formulated into vaccines in neutral or salt form. Pharmaceutically acceptable salts include acid addition salts (formed with the free amino groups of the peptide) and inorganic acids such as hydrochloric acid or phosphoric acid, or acetic acid, mineral acid, tartaric acid, mandelic acid, etc. Includes those formed by organic acids.

Salts formed with free carboxyl groups are, for example, from inorganic bases such as sodium, potassium, ammonium, calcium, or ferric hydroxide, and from isopropylamine, trimethylamine, 2-ethylaminoethanol, histidine, prohydroline, etc. It can also be derived from organic bases such as

Vaccines will be administered in a form compatible with the dosage formulation and in such amount as to be therapeutically effective and immunogenic. The amount administered depends on the subject being treated, the ability of the subject's immune system to synthesize antibodies, and the degree of protection desired. Precise amounts of active ingredient administered depend on the judgment of the practitioner and are peculiar to each individual. However, the suitable dosage range is −
It is on the order of several hundred micrograms of active ingredient per person.

Suitable dosages for initial administration and additional doses are variable, but typically the initial administration will be followed by subsequent injections or other administrations at one to two week intervals.

HTLV-III is known to undergo amino acid sequence variations, especially within the envelope gene [Starcich, B.
R, ) (1986) Cell 45 637-648
Page; Hahn, B, H,
1986) Science 232 1548-15
53], over 100 variants have been analyzed by molecular cloning and restriction enzyme recognition analysis, and some of these have also been analyzed by nucleotide sequencing. Some of these are RF [Po-povic
(1984) Science vol. 224, pp. 497-500], WMJ-1 [Hahn, B, H, et al. (1986) Sci.
nce Vol. 232, pp. 1548-1553], LAV [
Vain-Hobson φ varnish (Vain-Hobson,
(1985) Cel14G, pp.9-17]
and ARV-2C Sanchez-Pescador Earl (S
anchez-Pescador, R.) et al. (1
985) 5science Volume 227 484-492
Although the present invention describes sequences from one HTLV-m isolate, known as HTLV-m isolate, RIO, PBI, 590 and Any HTL
Appropriate envelope regions of V-III isolates can be created. HTLV-I from different virus isolates
The II protein can be used in vaccine formulations as demonstrated above to prevent infection by different ITLV-m isolates. Furthermore, the use of one recombinant antigenic protein from one or more HTLV-m isolates to create a vaccine formulation provides immunity against AIDS, providing even better protection.

EXAMPLES The following are examples illustrating the method of the invention, including the best mode. These examples are not to be considered limiting; unless otherwise noted, all proportions of solvent mixtures are by volume.

Example 1 Construction of plasmid pREV2.2 pREV2
．． A two-plasmid expression vector was constructed from plasmid pBGI. Plasmid pBG1 can be isolated from the E. coli host using well-known procedures, such as the clear lysate 1 isopycnic gradient method. Like pBGl, pREV2.2 expresses the inserted gene behind an E. coli promoter. The difference between pBGl and ρREV2.2 is as follows.

1. pREV2.2 lacks functional replication of plasmid (rop) proteins.

2.11REV2.2 has the trpA transcription terminator inserted at the AatII position, this sequence ensures transcription termination of overexpressed genes.

3. pREV2.2 has genes that provide resistance to ampicillin and chloramphenicol;
BGl provides resistance to ampicillin only.

4. pREV2.2 contains sequences encoding several restriction enzyme positions.

The following procedure, shown in Figure 1 of the drawings, was used to generate each of the four differences listed above.

Ia, plasmid pBG1 (5ug) was transferred to Nde
Restriction with I yielded two fragments of approximately 2160 and 3440 base pairs.

lb, D from the digestion mixture after inactivation of Ndel
NA0.1ag was treated with T under conditions favorable for intramolecular recombination.
4 DNA ligase using standard T4 ligase reaction conditions, reaction volume 200μ+) [New England Biolabs, Beverly, MA].

Intramolecular recombination of the 3440 base pair fragment resulted in an ampicillin resistant plasmid. The recombinant mixture was transformed into the recipient strain IWJ Old 03 (obtained from New England Biolabs) and ampicillin resistant clones were selected by standard procedures.

lc, the product plasmid pBG1ΔN when the 2160 base pair Nde I fragment was deleted from pBGl is
A plasmid was created from an ampicillin-resistant clone, and N
The product fragments selected by determining the restriction digestion pattern with deI and SalI were approximately 1790 and 1
650), this deletion allows r to control plasmid replication.
The Op gene is inactivated.

2a, pBGIΔN (5 μg) was then digested with EcoRI and Be11 and the larger fragment of approximately 2455 base pairs was isolated. 2b, a double-stranded synthetic fragment with the structure shown in Table 1 was prepared by Itakura et al.
a, ni,), Lodge Jay (Rossi,
, 1-J,) and Wallace R.B.
ace, R, B, Xl984) Ann.

Rev, Bioches, Vol. 53, pp. 323-356.
and references therein]. This fragment has sticky ends of eel I and EcoRI and contains recognition sequences for several restriction enzymes.

2c, 2455 base pair EcoRI-eel I
0.1 ag of the fragment and 0.01 μg of the synthetic fragment were combined with T40NA ligase, and competent cells of strain J old 03 were transformed.

Cells that received the recombinant plasmid in which the synthetic fragment was inserted between the BclI and EcoR1 positions of p8GIΔN were selected by digesting the plasmid with Hpa I and EcoRI. The size of the diagnostic fragment was approximately 235.
5 and 200 base pairs. This plasmid is pREV
It is called l.

2d, pREVl (>5 μg) undergoes specific cleavage when digested with AatII.

2e, the double-stranded fragments shown in Table 2 were synthesized. This fragment has Aat U sticky ends and contains the trpA transcription termination sequence.

2f, pREVl (0,1
ag) was recombined with 1 μg of synthetic fragment 0, O in a volume of 20 μm using T4 DNA ligase.

2g, cells of the competent strain JM103 were transformed and ampicillin-resistant clones were selected.

2 h, double restriction digests with pnr and EcoRI of plasmids isolated from selected colonies were used to isolate cells containing the correct structure. Kpni and E
The size of the coRI generated fragment is approximately 2475 and 80 base pairs. This plasmid is called pREVITT,
Contains the trpA transcription terminator.

3a, pREVITT (5 ug) prepared as above (standard methods) was incubated with Nde I and Xmn1
A fragment of approximately 850 base pairs was isolated.

3b, 5 μg of plasmid pBR325 (BRL, Gaithersburg, MD) containing genes conferring resistance to chloramphenicol and ampicillin and tetracycline was cleaved with Bcl I and the ends were engineered with Klenov polymerase and deoxynucleotides. . After inactivating the enzyme, the mixture was
A fragment of approximately 3185 base pairs was isolated. This fragment contains the genes for chloramphenicol and ampicillin resistance and the origin of replication.

3c, Nde I-Xmn from pREVITT
0.1 gg of I fragment and Nde I from pBR325
-eel I fragment and 20 μl using 74 DNA ligase.
The mixture was used to transform competent cells of strain JO3. Cells resistant to both ampicillin and chloramphenicol were selected.

3d, plasmids from selected clones were transformed into Ec
A double digest was performed with oRI and Nde I and this digest was used to select for plasmids that yielded fragment sizes of approximately 2480.1145 and 410 base pairs. This is plasmid p
It is called REVITT/C old and has a gene that is resistant to both ampicillin and chloramphenicol.

4a, the double-stranded fragments shown in Table 3 were synthesized. This fragment has a plant end and an Sst I sticky end and contains recognition sequences for several restriction enzyme positions. 4b
, pREVITT/chi (5 μg) was injected with Nru I
(cleaves approximately 20 nucleotides from the Bcl 1 position) and 5stl (cleaves within multiple cloning positions). The larger one, approximately 3990 base pairs, was isolated from a 7 galose gel.

4c, Nru I from pREVITT/chl
-5st I fragment 0.1 #8 and synthetic fragment 0.01ag
was treated with T4 DNA ligase in a 20 μm volume.

4d. This mixture was transformed into strain Jold03 and ampicillin-resistant clones were selected.

4e. Purify the plasmid from several clones,
Selection was made by digestion with old uI or ClaI. Recombinant clones with new multiple cloning positions will yield a single fragment when digested with either of these enzymes, as both cleave the plasmid only once.

4f, Sequences of multiple cloning positions were verified.

To do this, combine the plasmids with Hpa I and Pvu■
and isolated a 1395 base pair fragment, which was mp
18 into the Sea 1 position and its sequence determined by dideoxynucleotide sequencing using standard methods.

This plasmid, designated pREV2.2, is illustrated in Figure 2 of the drawings.

Example 2 Construction of pRlo and its expression plasmid ρR
IO consists of about 12 pieces of DNA that essentially encodes the HTLV-m env gene from the pn1 position to the Bgl■ position.
It contains 75 base pairs, from which a fusion protein of approximately 95 kn containing this portion of the gp120 envelope protein is synthesized. This plasmid can be constructed as follows.

1. Synthesize DNA having the sequence shown in Table 4. This D
The NA fragment can be synthesized by standard methods [Itakura et al., supra, and references therein] and encodes a portion of gp120. It has a plant terminus at 59 and a terminus at the 3' end that recombines with the BamHI overhang.

2. Restrict with plasmid ρBG15μglt act I, fill in the overhang ends with Klenov polymerase and deoxyribonucleotide triphosphates (dNTPs), restrict the fragment with BamHI, and isolate the large fragment of approximately 8.9 kb from an agarose gel. .

3. Using T4 DNA ligase, fragment 0 in Table 4
．． 1ag was recombined with pBG1 fragment 0.1ag in a 20μI volume and the recombination mixture was added to competent cell line 5G.
20251 [Gottesman Nis
n, S,), Halpern, E.
) and Tris-Ier, P.
) (1981) Journal of Bacter
14B, pages 267-273], and ampicillin-resistant transformants are selected.

4. The AhaI[I restriction pattern of the purified plasmid is used to select cells that have received the recombinant plasmid with the synthetic fragment, which has the plant end of the fragment in the Bc order supplemented with the ρBGI fragment. end-to-end recombined,
The Bas+HI overhangs are oriented so that they are reattached together. Digesting the appropriate plasmid with Aham yields approximately 5300.3170.690.640.3
Fragment lengths of 30, and 20 base pairs are obtained. 5.
50μ8)■
2x medium containing 1 part ampicillin [22 yeast extract, bactodributone, casamino acids (Difco, Detroit, MI), 0.2 x-basic potassium, 0. When the total amount of cellular protein was electrophoresed on a 5DS-polyacrylamide gel, approximately 95 kn of the dominant protein was detected by Coomassie Blue staining or AIDS, A.R.
can be visualized by Western blot analysis using selected sera from individuals infected with C. or HTLV-III as probes.

Example 3 ITLV-II (from plasmid ρRIO)
Purification of Recombinant Protein Containing the I Envelope Sequence 1. Growth of 11 Cells Cells were grown in a 10 liter volume in 2x medium in a Cheemup fermenter (Cheemup Inc., Woodbury, NY) at a fermentation temperature of 37°C. , pHa, s, and air were supplied at lvvm. Plasmid selection was performed using 50ag/
+gl ampicillin provided. Typical cell yield (
wet weight) was 30 g/I.

2. Cell lysis of E. coli 508 (wet cells [1] containing recombinant HTLV-III envelope fusion protein, 50IIM Tris-MCI (pH 8,0), 5+++M4 ethylenediaminetetraacetic acid (EDTA), 5mM dithiothreitol (DTT), 15mM β-mercapto X9/-
0.5X TRITON RX-100, and 5mM phenylmethylsulfonyl fluoride (PMSF) in a final volume of 100ml. 300 mg of lysozyme was added and the suspension was incubated at room temperature for 30 minutes.

This material was lysed using BEAD-BEATER™ (Biospec Products, Inc., Purdleville, Okla.) containing the same amount of 0.1-0.15 micron glass beads. Bacteriolysis was performed at room temperature for 6 days at 1 minute intervals. The liquid was separated from the beads and centrifuged at 20.000xg for 2.5 hours. Remove the supernatant and add Pelle'7
) to 8M urea, 20s+M) Lis-HCI (pH 8,
0), 5mM DTr.

15s+Mβ-mercaptoethanol, 5mM PMS
Dissolved in 100 ml consisting of F and 11 EDTA.

The pellet was solubilized using a Polytron homogenizer (Beckman, Berkeley, CA).
Centrifugation was performed at 20°,000×g for 2 hours.

3. Diethylaminoethyl (DEAE) chromatography DEAE Fast Flow 5EPHARO5E”
(Pharmacia, New Jersey), filled with 8 urea, 20+wM) Lis-) 1c
The supernatant was loaded at room temperature onto a 550-1 column (5 c x 28 c-) equilibrated with I(ρ)l 8.0), l 5 + 1 M β-mercaptoethanol, and ImM E[lTA. The column was washed with 1.5 liters of equilibration buffer and the protein was washed with 0-0.8M NaCl in the equilibration buffer.
It was eluted with a 5.0 liter linear gradient. HTLV-m
Proteins were eluted with 0°2M NaCl and 5DS-
Polyacrylamide electrophoresis was used to track and assay the dominant protein of approximately 95 kO.

Fractions containing HTLV-m protein were combined using a stressed cell positive pressure concentrator (Amicon, Danvers, MA) fitted with a molecular weight IO,000 cutoff membrane (YM-10, Amicon). Protein was concentrated to 10 ml. Filled with Super Fine Sephacryl S-300 (Pharmacia), 8M
Urea, 20mM Tris-HCI (pH 8,0), 15
s+Mβ-mercaptoethanol, and 1mM EDT
5501 column (2,5 cs x 10
0 cm) was loaded with the concentrate. The column was eluted with equilibration buffer at room temperature in 0.5 ml (maintaining a D flow rate.
eluted at %.

Example 4 Construction and expression of plasmid pP81 ++
It contains approximately 540 base pairs of HTLV-II[env gene [lNA] up to the gp
This part of the r20 envelope protein contains approximately 2
A 6 kD fusion protein is synthesized from this. This plasmid can be constructed as follows.

1. Synthesize DNA having the sequence shown in Table 15. This ON
The A fragment can be synthesized using standard methods and encodes part of gp120. It has a plant end at the 5' end and 3
It has an end that recombines with the BamHI overhang at the ' end.

2. Plasmid pREV2.2 (5 μg) EcoR
The large fragment of approximately 4 kD is isolated from an agarose gel.

3. Using T4 DNA ligase, fragment 0.1a glt 20a lcD capacity TepREV2.
Recombine with 0.1 μg of 2M fragment, transform the recombination mixture into competent cell strain 5620251, and select ampicillin resistant transformants.

4. Using the A'haIII restriction pattern of the purified plasmid, select cells that have accepted the recombinant plasmid with the synthetic fragment, where the plant end of the fragment is recombined with the EcoRV end of pREV2.2. and B
The amHI overhang ends are oriented so that they are reattached together. When a suitable plasmid is digested with Aha■, approximately 1210.1020.750.690.500.
Fragment lengths of 340 and 20 base pairs are obtained. A strain that has received this recombinant plasmid is grown in 2z medium containing 50 ag/s+l of ambicillin, and the total amount of cellular protein is electrophoresed on an S[)S-polyacrylamide gel, resulting in approximately 26 kD of protein. Proteins were stained with Coomassie blue or AIDS, ARC or HTLV-
can be visualized by Western blot analysis using as probes selected sera from individuals infected with M.

Example 5 7 Lasmi F pPB1+zskara+7)
Purification of Recombinant Protein Containing the HTLV-III M Bellobe Sequence 1. Cell Growth Cells in 21 medium were grown in a 10 liter volume in a Cheap fermenter at a salinity of 37°C, pH 6,8, and air. 1 vv+m. Plasmid selection is
Supplied with 50μ811 ampicillin and 20μg/ml chloramphenicol. Typical cell yield (wet weight) was 30 g/l.

2. 50 g (wet cell weight) of E. coli containing 1 ill @ lytic recombinant I (TLV-III envelope fusion protein) was added to 501 M
) Lis-ICI (pH 8,0), 5% H ethylenediaminetetraacetic acid (El)TA), 5+mM dithiothreitol (DTT), 15n+Mβ mercaptoethanol, 0.5χTRITON"X-100, and 5mM phenylmethylsulfonyl fluoride ( resuspended in a final volume of 100 l of a solution consisting of
0 B was added and the suspension was incubated for 30 min at room temperature.

This material was lysed using a BEAD-BEATER'' (Biospec Products, Inc., Puddle Spill, Okla.) containing the same amount of 0.1-0.15 μm glass beads. Lysis was performed for 6 minutes at 1 minute intervals at room temperature. Separate the liquid from the beads at 20,000λ
Centrifuged at g for 265 hours. The supernatant was removed and the pellet was treated with 6M guanidine hydrochloride, 20mM) Lis-II.
CI (1188,0), 5mM DTT, 15*
Dissolved in Mβ-mercaptoethanol, 5n+M PMSF and 1mM EDTA (100a+I). Solubilize the pellet using a Polytron homogenizer and
Centrifuged at 0.000xg for 2 hours.

The supernatant (90 ml) was mixed with 8 M urea, 20 mM potassium phosphate (pn 7.0), 1 a+M EDTA, and 1
Dialyzed against 5 mM β-mercaptoethanol (4 liters). Dialysis was performed using Spectrafer dialysis tubing (S/P, McGraw-Hill, IL) with a cutoff of 0 molecular weight, 3.5 kD, and the buffer was changed three times for at least 8 hours each time.

3. CM chromatography CM Fast Flow Sepharose (Fast Fl
owSEPHARO5E”) (Pharmacia), 8M urea, lsM potassium phosphate (pH 7,0
), 15 intestinal Hβ-mercaptoethanol, and ImMε
550111 column (5cs+ x
28 cm) was loaded with dialysate at room temperature.

Wash the column with 2 liters of equilibration buffer to remove the protein.
- Eluted with a 5.0 liter linear gradient of 0.4M NaCl. HTLV-111 protein (26 in) is 50
When assayed by S-polyacrylamide gel electrophoresis,
Eluted at approximately 0.2M NaCl.

Example 6 Construction and Expression of Plasmid p590 Plasmid p590 contains approximately 1055 base pairs of DNA essentially encoding the ITLV III env gene from the PvuII position to the old ndI [1 position], and contains gp160
This part of the envelope protein contains about 86 k
The D fusion protein will now be synthesized. This plasmid can be constructed as follows.

(2) Synthesize DNA having the ψ sequence shown in Table 6. this DNA
The fragment can be synthesized using standard methods and encodes part of gpteo. It has a plant end at the 5' end and a 3'
It has an end that recombines with the old ndnI overhang.

2. Convert plasmid pREV2.2 (5Mg) into EcoR
V and ) Iindm and the large fragment of approximately 4 k[+ is isolated from an agarose gel.

3. Using T4 DNA ligase, fragment 0 in Table 6
．． 1 Mg is recombined with 0.1 μg of pREV2.2 fragment in a volume of 20 ml (7) and the recombinant mixture is transformed into competent cell strain 5G20251 to select for ampicillin resistant transformants.

4. Using the AhaIII restriction pattern of the purified plasmid, select cells that have received a silk recombinant plasmid with a synthetic fragment in which the plant end of the fragment is religated to the EcoRV end of pREV2.2; )I
The indm overhanging ends are oriented such that they are reattached together. When a suitable plasmid is digested with Aha■, approximately 1740.1020.750.690.500.
Fragment lengths of 340 and 20 base pairs are obtained.

5. 5 μg of plasmid purified from this strain was injected with Nd
Limit by e I and S+ga I. A fragment of approximately 1425 base pairs is isolated from an agarose gel. A 1505 base pair fragment is fused to the DNA encoding the gpteo fragment.

6. Restrict the plasmid ρBGIOI with BamHI, fill in the overhanging ends with Klenov polymerase and dNTPs, and then restrict the fragment with NdeI. Approximately 6.5
The kO fragment is isolated from the agarose gel.

? , using T4 DNA ligase, Nde I-
5+wa I fragment 0.1M to pBG+fragment 0.1M
The recombination mixture is transformed into competent cell strain 5G20251 and ampicillin-resistant transformants are selected.

8. Using the Ahanl restriction pattern of the purified plasmid, select cells that have received the recombinant plasmid with a synthetic fragment that recombines the Sma I plant end of the fragment to the supplemented BamHI end. and N
The de I overhanging ends are oriented so that they are reattached together. Digestion of the appropriate plasmid with AhaII yields a fragment length of approximately 5900.1020.690.430 and 20 base pairs.

9. The bacterial strain that received this recombinant plasmid was treated with 50 Mg
When grown in 2z medium containing empicillin and electrophoresing the total amount of cellular protein on a 5DS-polyacrylamide gel, a protein of approximately 86 kD was detected by Coomassie blue staining or AIDS, ARC, or HTLV- can be visualized by Western φ plot analysis using as probes selected sera from individuals infected with M.

Example 7 Purification of Recombinant Protein Containing HTLV-m Envelope Sequences from Plasmid p590 1. Growth of Cells Cells were grown at 10+7 tttle volume in 2x medium in a Cheap fermenter at 0 fermentation temperature 37°C, pH 6. ,8,
and air was supplied at lvv■. Plasmid selection is
Provided with 50μ8/-1 ampicillin. Typical cell yield (wet weight) was 30 g/I.

2. Cell lysis of Bacillus enterica 508 (wet cell weight) containing recombinant HTLV-m envelope fusion protein, 5 (1 wM) Lys-MCI (PH8,0), 5a+M EDTA, 5
mM DTT, 15mM β-mercaptoethanol, 0.5X TRITONllX-100, and 511
Resuspend in a final volume of 100 l of MPMSF. 300 a+g of lysozyme was added and the suspension was incubated at room temperature for 30 minutes.

This material was lysed using BEAD-HEATER™ containing the same amount of 0.1-0.15 av+ glass beads.

Lysis was performed for 6 minutes at 1 minute intervals at room temperature. The liquid was separated from the beads and centrifuged at 20.000xg for 2.5 hours. The supernatant was removed and the pellet was washed with 6M guanidine hydrochloride, 20mM) Lis-HCI (pH 8,0), 5
1M DTT.

15 Goa β Mercaptoethanol, 5MM PMSF
and resuspended in IIIMEDTA (100w+I).

The pellets were solubilized using a Polytron homogenizer and centrifuged at 20,000xg for 2 hours.

The supernatant (901) was mixed with 8M urea, 20mM) Lis-HC.
I(+)H8,O), 1 mM EDTA, and 15
Dialyzed against mM β-mercaptoethanol (4 liters). Dialysis was carried out by changing the buffer three times, each time changing the buffer solution 8 times.
It took more than an hour.

3. Diethylaminoethyl (DEAE) chromatography DEAE Fast Flow Sepharose (5EPHAROSE”) (Pharmacia)
urea, 20a+M tris-HCI (pH 8
,0), 15mM β-mercaptoethanol, and Im
550111 column (5 cm) equilibrated with M EDTA
x 2B cm+) was loaded with dialysate at room temperature.

The column was washed with 1.5 liter equilibration buffer and the protein was eluted with a 5 liter linear gradient of 0-0.8M NaCl in buffer. HTLV-III protein is approximately 0.
It was eluted with 4M NaCl and assayed using 5O5-polyacrylamide gel electrophoresis, tracking the dominant protein of approximately 86 kO.

The fractions containing the HTLV-t[I protein were combined and separated with a molecular weight of 1O9000.
The protein was concentrated to 10 ml using a stress cell positive pressure concentrator (Amicon) fitted with a M-10 (Amicon).

Super Fine 5EP) IACRYLR5-300 (
Pharmacia), 8M urea, 20ml)
50 equilibrated with Lis-○CI (11H8,0), 15mM β-mercaptoethanol, and 1mM EDTA.
A 01 column (2,5 cm x 100 cm) was loaded with the concentrate. The column was eluted with equilibration buffer at room temperature. A flow rate of 0.51 per minute was maintained. HTLV-■ protein eluted at approximately 40X column volume.

Example 8 Old construction and expression of plasmid p into plasmid p
KH1 essentially contains approximately 1830 base pairs of DNA encoding the HTLV-III env gene from the pnI position to the Hindu11 position, and contains approximately 70 kD that contains this portion of the gp160 envelope protein.
A fusion protein will be synthesized from this. This plasmid can be constructed as follows.

1. Synthesize DNA having the sequence shown in Table 7. This [lN
Fragment A can be synthesized using standard methods and encodes part of gp160. It has a plant end at the 5' end and 3
It has an end that recombines with the Hindm overhang at the ' end.

2.1 The lasmid pREV2.2 (5g g) was restricted with old ul, the DNA was treated with Klenov polymerase to plant the ends, and then treated with old ndI[I to create a large fragment of approximately 5 kD. Fragments are isolated from a 7 galose gel.

3. Using T4 DNA ligase, fragment 0 in Table 7
．． 1 gg of pREV2.2 fragment 0.1 in 20 μm volume
The recombination mixture is transformed into competent cell strain CAG629 and ampicillin resistant transformants are selected.

4. Using the Aha [[ restriction pattern of the purified plasmid, select for cells that have accepted the silk recombinant plasmid with the synthetic fragment, where the plant end of the fragment is reattached to the old ul end of ρREV2.2. combined and former ndnl
The overhanging ends are oriented such that they are reattached together. When a suitable plasmid is digested with Aha■, approximately 1
730, +020.750.690.640.600.
Fragment lengths of 340 and 20 base pairs are obtained. A strain that has received this recombinant plasmid is grown in 2z medium containing 50 μg/111 ampicillin, and when the total amount of cell proteins is subjected to electrophoresis on a 5DS-polyacrylamide gel, approximately 70 kO of protein is extracted using Coomassie blue. by staining or AIDS, ARC or HTLV-nI
can be visualized by Western blot analysis using selected sera from individuals infected with the virus as probes.

Example 9 Purification of a Recombinant Protein Containing the Classic HTLV-m Envelope Sequence in Plasmid p ■ Growth of the Cells Cells were grown in a 10 liter volume in 2x medium in a Cheap fermenter at a fermentation temperature of 32°C. , pH 6,8, and air at 1 vv+g. Plasmid selection is
Provided with 50 μg/ml ampicillin. Typical cell yield (wet weight) was 30 g/I.

2. Cell lysis 50 g (wet cell weight) of Escherichia coli containing the recombinant silk HTLV-m envelope fusion protein was treated with 50 IIM Tris-HCI (pH 8,0), 5+w EDTA, 5+wM dithiothreitol (DTT), 15mM/13-mercaptoethanol. ITON' X-100 and 5+m
Resuspended in a final volume of 100 ml of solution consisting of M PMSF. 1 m8 of Lysozyme 300 was added and the suspension was incubated at room temperature for 30 minutes.

This material was lysed using BEA [1-BEATERTM (Biospec Fist Products) containing the same amount of 0.1-0.15 μm glass beads. At room temperature,
Bacteriolysis was performed for 6 minutes at 1 minute intervals. The liquid was separated from the beads and centrifuged at 20,000x8 for 2.5 hours.
Remove the supernatant and add the pellet to 8M urea, 20w+M
) Lis-■CI (pH 8.0), 5s+M [lTT,
15mM β-mercaptoethanol, 5M PMS
F and 1mM EDTA (100+wl).

The pellet was solubilized using a Polytron homogenizer (Beckman, Berkeley, CA) and
Centrifuged at 0.000xg for 2 hours.

3. DEAE Chromatography DEAE Fast Flow SEPHAROSE”
(Pharmacia), 8M urea, 20n+M
) Lis-HCI (pH 8.0), 15mM β-mercaptoethanol, and 1mM EDTA.
The supernatant was loaded onto a 501 column (5 cm x 28 cm) at room temperature. The column was washed with 1.5 liters of equilibration buffer and the proteins were washed with O-0.8M NaCl in buffer.
It was eluted with a 5 liter linear gradient of . The HTLV ■ tank was eluted with approximately 0.2M NaCl and assayed using 5DS-polyacrylamide gel electrophoresis to track protein at approximately 70 kO.

The fractions containing HTLV-III protein were combined and separated using a 10,000 molecular weight cut-off membrane (VM-t
The protein was concentrated to 10 ml using a stress cell positive pressure concentrator (Amicon, Inc.) fitted with a 1000 ml tube (Amicon, Inc.).

Super Fine 5EPHACRYL” S-300 (
5001 column (2.5 cm x
100 cm) was loaded with the concentrate. The column was eluted with equilibration buffer at room temperature. The flow rate was maintained at 0.51/min, )ITLV-■ protein was approximately 40% of the column volume.
It was eluted with

4. Combine the Hl-containing fractions for SOS-polyacrylamide electrophoresis and perform a molecular weight of 10,000.
Proteins were concentrated using a stress cell positive pressure concentrator equipped with a cut-off membrane. 1 g of protein 2 was mixed with loading buffer and transferred to a M.W.
W. Hunkapiller), Yi Lujan (E
．． Lujan), F.Ostlander (F.Ost)
rander), and L.E. Food (L.E.
Hood) Method in Enzya+olo
Electrophoresis was performed through a preparative SDS-polyacrylamide gel (40 cm x 20 cts x 4 am) as described in J. Gy, Vol. 91, pp. 227-236 (1983). 70kD (D 11TLV-
111 was visualized with 0.25M MCI and eluted from the gel as described above. Proteins can be removed from SDS by precipitating them with acetone [Dynan W.
Dynan.lj.J., Jendrisak, J.

J. ), Hager φ Day Association (Hager. 0.
A. ) and Burgess R.
s, R. R. ) (1981) J. Biol. C
hem. Volume 256, pages 5860-5865.

Example 10 Construction of a non-fusion derivative of PBI Non-HTLV other than N-terminal methionine ■P containing no amino acids
Non-fusion derivatives of BI proteins can be produced by oligonucleotide-directed site-directed mutagenesis [Inouuni Nis, Inouuni
Mr DNA and RNA synthesis and applications J (Synt
hesis & Applications of D
NA & RHA), Narang φ Saran A, ed., Academic Press, 1987].
This procedure involves translating the env gene sequence of pPBl into a non-H T L V-I [190 bp upstream and a 39 bp downstream
was deleted via DNA loop-out caused by hybridization with synthetic oligonucleotides.

The oligonucleotide synthesized for the N-terminal loopout is designed so that the start codon of the β-glucuronidase gene is placed adjacent to the 5' end of the HTLV<em>env gene sequence (Figure 4). .

The oligonucleotide contains sequences homologous to both sides of this newly created junction, allowing proper hybridization to plasmid DNA.

The two DNA molecules used to form the heteroduplex with the single-stranded gap substrate for hybridization were made by digesting ppet with Sall and Hpa 1 or with Pst I alone. Linearized p
pe I was digested with Pst I, and Sal I and Hp
Double digestion with a [ generated 3800 and TOO bp fragments, the larger of which was gel isolated for mutagenesis.

Kinase treatment of oligonucleotides, hybridization, polymerization, and recombination to create closed-circle molecules were performed according to the method of Inouni and Inouni, supra. To enrich for DNA molecules containing the deletion, the DNA mixture was digested with old UI, which made cuts within the region to be deleted.

Using digested DNA, competent E. coli 3M1
05 cells were transformed with VT (8 g tryptone, 5 g yeast extract, and 5 g NaCl per liter)C
Transformants containing the plasmid were isolated by growing overnight at 37° C. on m plates.

Plasmid DNA was isolated from each transformant and screened for correct structure by co-digestion with old UI and Hindm. The molecules that were not deleted were approximately 3900 and 600.
A bp fragment was generated. The molecule containing the deletion is the old u1
It had no position and produced a linear molecule of about 4400 bp.
Plasmid DNA from transformants suspected of containing deletions
A was retransformed to ensure separation of deletion and deletion-free plasmids and recovery of a pure plasmid population. DNA from these second transformants was analyzed as in the previous digest and determined to have the correct structure. This plasmid was designated pΔPBI.

To eliminate the C-terminal non-HTLV-III amino acids, oligonucleotide-directed site-directed mutagenesis was performed using the pΔPBI plasmid as a substrate. The oligonucleotide (Figure 5) positions the TGA codon so that it occurs downstream from the env gene sequence when out of frame, immediately adjacent to the 3' end of the env gene sequence, and as a translation stop codon when in frame. intended to work.

The heteroduplex-forming molecules used for hybridization include pΔPBI with Pstl alone or K
It was prepared by digestion with pn I and Hpa I. A large KpnI/)Ipa that encompasses most of the vector
The I fragment was gel isolated for mutagenesis. Kinase treatment,
Hybridization, polymerization, and recombination were performed as above. Enrichment of deleted molecules was achieved by digestion with old ndm that made cuts within the region to be deleted. 0 DNA was used for cell transformation as above.

Plasmid [lNA was isolated from each transformant and Ec.

Correct structure was checked by co-digestion with R1 and Hpa I. Deletion plasmids are 2900 and 1750
Plasmid DNA exhibiting this pattern, resulting in two restriction fragments of bp, was retransformed as above, and DNA from these transformants was analyzed in the same digestion. N-
This plasmid, containing terminal and C-terminal deletions, p
It is designated as d2PB1.

The strain that received the plasmid pΔPal was grown at 50μ3/m
2z medium [2% yeast extract, Bactodributon, Casamino acidic Difco, Detroit, Michigan] containing 1 ml of ampicillin, 0.2 nibase potassium, 0.2 x
When grown in dibasic potassium, and 0.2x dibasic sodium] and subjected to electrophoresis of the total amount of cellular protein on a 5O5-polyacrylamide gel, approximately 22 kD of protein was detected by Coomassie blue staining or the recoated env gene. It can be visualized by Western blot analysis using sera selected from animals immunized with the protein as probes. Under the same conditions approximately 20 kO of protein is produced in the strain containing pd2PB1.

Oligonucleotide-directed site-directed mutagenesis techniques can be used in a similar manner to bypass the enV gene fusion protein RIO1590, and to eliminate non-HTLV-nl amino acids.

In the above procedure, non-HTLV-III from the fusion protein
Sequence removal involves removing amino acids at both the N-terminus and C-terminus of the protein and is accomplished in two consecutive steps.

N-end! It is well known in the art that methionine at the 4-position is enzymatically cleaved using the enzyme methionine aminopeptidase (MAP). MAP is E. coli [Ben:
Hen-Bassat, A., Bauer, Ni., Chan Nis.
((hang.

S, Y,), Myambo Kei (Myambo, Ni,),
Booss+an, A. and Chap, S. (1987) J.
of Bacteriol, Volume 169 (No. 2) 75
1-757] and Salmonella typhimurium (Salsonell)
a typhimurius+),
In vitro activity was demonstrated for transcolloid proteins [Miller, C.

G,), Strauch+, L,), Kukral ΦM,
A.M.), Miller, J.L.
J, L, ), Wingfield Temple and - Te4-' (W
ingfield, P, T,), Massei, G, J-, Werlen, R, C, Graber, P, and Mov7” N.R. (Movva +N, R,) (1987) Proc.
, Natl, Acad, Sci, USA, Vol. 84, pp. 2718-2722], therefore, N-terminal methionine removal can occur in vivo when the MAP-producing host (e.g.
This can be accomplished by expressing the protein in bacteria I (CM89 or S. cerevisiae) or in vitro using purified MAP (eg, as per the procedure of Miller et al.).

Purification of pd2PB1 Unless otherwise specified, all steps are performed at room temperature.

[Thaw 3 bottles of frozen cell paste containing 1 liter of pd2PBl at 37°C, JS-4,2
Placed in a J-68 centrifuge (Beckman, Palo Alto, Calif.) with a 0-temperature filter at 4°C, LOGO rpm.
Rotate with + for 30 minutes. Discard the supernatant and measure the cell pellet weight. Cell pellet (typically 1 kg)
8 to 1 urea, 20 mM)'/su-HCI (pH
7,5±0.1), 1mM EDTA, 14.7a+
Resuspend in 2 volumes of lysis buffer (v/w) consisting of M2-mercaptoethanol and 1mM PMSF.

0.5-0.7 s ■ TDK type pilot DYNO-former LL containing glass beads (Inpandex,
Maywood, New Chassis), re! Prior to use, pass the J-turbid cell pellet through D'/NO-MILLJZ lysis W! at 200-400 ml per minute. 1ljff
Charge 1 liter and cool the apparatus so that the solution runs below ambient temperature. D the resuspended cell pellet
Pass through “YNO-old LL” twice, and after the second pass, DYN
Wash the O-old LL''' with 1αl of lysate a. Save the lysed cell suspension and washing solution together.

Concentration and Filtration - The lysed cell suspension plus 1 liter of wash solution was purified using a 0.45μ DtJRAPORE™ Pellicon cassette in a Pellicon 4 GPM system (Millipore, Bedford, MA). Concentrate to 8001* Concentrate at an artificial pressure of less than 40 psI and an outlet pressure of IO to 20 psi.

After concentration, the lysed cell suspension is filtered with 4 liters of lysis buffer using the same pericon system, cassette, and pressure settings fitted with diafiltration tubing.

[Extraction] - The washed lysed cell suspension was extracted with 6M guanidine HCI, +
00a+M)Lis-)ICI (pH7,6±0.1)
, and 10mM EDTA.
Extracted in liters.

[Buffer exchange]-PTG (J sets of 2 (10,000
The filtrate from the previous step is typically concentrated to 1 liter using a Vericon 4GPM system with 0 NMML). 5
Concentration is carried out at a population pressure of less than 0 psi and an outlet pressure of 30 to 45 psi. After concentration, the supernatant is diluted with 8M urea, 25
mM potassium phosphate, and 1 mM EDTA (pH 6,
The buffer is exchanged with a CM column buffer consisting of 8±0.1) having a conductivity of 3,0-S/C- or less. For buffer exchange, the same Pellicon system with diafiltration tubing, the same cassette and the same pressure settings are used as described above.

Eight liters of CM column buffer is used to buffer exchange one liter of concentrated extract. After buffer exchange, extract the buffer-exchanged extract from the system, wash the system with 1 liter of ω column buffer, combine the buffer-exchanged extract and wash, and measure the conductivity and pH of the solution. The solution conductivity is adjusted to 3.0 ms/cm with deionized urea and the pH is adjusted within the range of 6.5-7.0.

[CM chromatography]-CM Sepharose (SEP
HARO5E') Fast 7c2- (FAST F
A 50 x 51 c-column (77-Macia, Bis., New Jersey) with 4 column volumes of 0.5 M NaOH.

2 column volumes of deionized water, and 2-3 column volumes of C
Equilibrate by successive washes with M column buffer. The pH of the effluent is within 0.2 units of the CM column buffer and the conductivity of the effluent is 0.3 + of the CM column buffer.
The column is considered to be in equilibrium when it is within *s/cm.

For loading, add 10 or more buffer exchanged extracts! 5
Pump into the column at an artificial pressure of psi. After loading, wash the CM column with CM column buffer until the optical density (00) at 280 ns+ of the effluent is less than 0.1. 8
Elute pd2PB1 with a little linear gradient, 100 1
Collect fractions. 505-PAG fraction
E. and Western with anti-gp160 antibody and fractions containing significant pd2PB1 and trace contaminants are combined.

[Organic extraction] - The combined protein solution from the previous step is brought to a ratio of 55 x acetonitrile to 45 x protein solution (V/V) by gradual addition of pure acetonitrile while stirring. was added and the solution was centrifuged to 10,000 ml in a J2-21 centrifuge using a JAIO rotor (Beckman).
Centrifuge for 15 minutes at rp-14°C.

After centrifugation, collect the supernatant and discard the pellet.

The centrifugation supernatant was diluted with ethanol 35× by gradual addition of pure 95× ethanol with stirring.
After adding all the ethanol, bringing the ratio of Z to supernatant to 65% (v/v), the solution was spun at 10,000 rpm in a J2-21 centrifuge using a JA-10 rotor.
, centrifuge for 15 minutes at 4°C. After centrifugation, collect the pellet and discard the supernatant.

The pellet was air-dried for 15 min and then treated with 8M urea, 0.3M glycine, 5sM EDTA, +5M2-mercaptoethanol, ImM dithiothreitol (DTT) (pH
Redissolve in S-300 column buffer consisting of 8,50±0.01). Dissolve the pellet in a volume of S-300 column buffer equal to one-tenth the volume of the protein solution stored at the beginning of this step.

[Concentration] - The absorbance of the redissolved protein solution from above is reduced to 28
Measured at 0n-, lmg/s+l solution of protein is 2
Measure the approximate protein concentration assuming an absorbance of 1.0 at 80 n-. The solution is concentrated to 10-g/ml using a 200-1 Amicon agitated cell concentrator equipped with a YM-10 membrane.

[S-300 Chromatography] - Concentrated protein solution 3
0.70■1 Cephacryl (5EPHACRYL”)
S-300 ()7-Macia) 5.0x135c
m column.

8M urea, 0.3M glycine, 5*M EDTA, 1
Equilibrate the column in advance with S.300 column buffer consisting of 55M 2-mercaptoethanol, Ig+M dithiothreitol ([1TT) (pHs, so±0.01).After loading, evenly coat the column with the same buffer. Manipulate. Collect 201 fractions, collect 5 fractions
The pd2P81 content is assayed by OS-PAGE.

Seven equal recoats of the appropriate fractions containing ρd2PB1 are taken and used to determine which fractions are satisfactory for pooling. Reserve aliquots, 8M urea, 25s+M sodium phosphate, 1 mM E D
Dialyze overnight against TA (pH 6,8±0.1) and measure the pH of the dialysed stock using the dialysis buffer as a blank. The protein 1 degree in solution is pd2PB1
is determined using the calculated extinction coefficient of 1.0(■g/+wl)-'.

5DS-PAGE is run on 10 μg of dialysis stock using a 15% 5057 acrylamide gel. After Coomassie staining and destaining, the gel is scanned using an LKB (Gaithersburg, MD) scanning densitometer connected to a Waters (Milford, MA) $1740 integrator.

If the pd2PB1 band on the gel is greater than 97% pure, the fractions used for aliquoting should be
lisulus amebocyte Lyz using a 6 eu/ml tube.
ate) (LAL) assay for bacterial endotoxins at 1- to 20-fold dilutions. If the LAL test on diluted fractions is negative, the fractions are pooled and used for subsequent manipulations.

If the gel does not meet purity specifications, repeat the method using equal aliquots from different sets of fractions, pooling only fractions with negative LAL tests at 1-20 fold dilutions.

ProValGluThrProThrArgGlu 1(,SerLeuAspArgGluAsnCysG
1ylleΣ) AsnValTrpAlaThrHi
sAlaCysVa co/// Shihiro'''LysLeuThrProLeuCysValS
erLetzun\ [AsnThrAsnSerSerSerGly
ArgMetA, CysSerPheAsnIleSerTh
rSer 工１eLeuThrSerCysAsnThr
SerValIlcGluPrO Engineering 1ePro Engineering 1eH
1sTyrCySAljMehiLeuArg :LysLysLeuAspG1yLeuTrpAla
Phe=AspGlnPheProValTrpLys
GluAla)AlaLySAla'ryrAspTh
rG1uValH1sLProThrAspProAS
nProG1nGluVal:AsnMet:TrpL
ysAsnAspMet, ValGluITrpAsp
GlnserLeuLysProcysValxLys
cysTh rAspLeuLysAsnAspTh
r:MetGluLysGlyGlu 1eLy
sAsn: ArgGlyl, ysValGlnLysG
luTyrAla)IleAspAsnAspThrT
hrSerTyrThr:ThrG1nAlacysP
roLysValserPheIProA1aGlyP
heAla Engineering 1eLeuLyscysATG(3TG ding(3GAAG(3AAGCAA(″″G
ATACAGAG (3TACA ding A/'t-
AACCCACAAGAAGTAGcon
= A t) TG A C A T G G T
A G A A C Go to 1 ACT^CCA
GCTATACGTT,, [: C A A r1G G T A T C
C T T T G i'” GCGATTC
TAAAATGTAtGTC^GCACAGTACAA
TC:CACCACTCTATDGTGCATCAGA
TGCT＾AAGCATAT”4TG dingTTGG[3C
CACACA Ding GCCTG Ding GTACCCACAGAC
CCC “ATTGGTAAATGTGACAGAAAA
TT Ding TAACA Ding G Ding GGAAA\GATGCATG
AGGA DingATAATCAGTTTATGGGATCA
AAGC＾DingTAACCCCACTCTGTGTDingAG
TTTAAA[;TGCACT(3ATTACCAADGAGTAGCGGGAGAATGATAATGG
AGAAAACTCTTTCAATATCAGCACAA
GCATA White G/A10GTA＾GGTGTTTTTA
TAAAACTTGATATAATATACCAATAGAT
AATGAT'Gt) C Yashiro GTTGTAACACCT
CA(iTcATTAcAcAGGcr:TGT+G
C C A White TTCCCATACATTATTGTG
CCCCGGCTGGTTTT+TAATAAG^CG
TTCAATGGAACAGGACC＾TGT＾C＾＾
White TiTAc White CATGGAATTAGGCCAGTA
GTATCAACTCAACTGCTGTT^AATG
GCAGTCTGGCAGA1 ＾G A C A
A T G C T A A^^CC^T^^TT G
T t＞CselfAGACCCAAACAACAAT^C
GGGAGhaGCATTTGDing TACAATAGG. A
ACATTA(3TAGAGCAAAATGOAAAG
AGAACAATTTGGAAr'lTA8T^81G
ACCCAGAAATTGTAACGCACAG^AT
TCAACACAACTGTTT＾HakuchoAGGCiGT
CA+KomaTAACACTGAA'GGAAG'CA^A
TTATAAAACATGTGGCAGGA+80TOG
ACAAATTAGATGTTCATC+C;G TC
; GTAA ding AGCAACAATGAGTC (AGAA
GAGGTAGTAATTAGATCTGCCAATT
TCAGTACAGCTGAACCCAATCTGTAG
AAATTAATAAGAAAAAAGTATCCGTA
TCCAGAGAGGGACCAAAAAAATAGGAA
ATATATGAGACAAGCACATGTTAHACA
CTTTAAAACAGATAGATAGCAAAATT
^White ACAATAATCTTTAA (3CAGTCCT
CAGGAGGGGTdingTTAATTGTGGAGGGGG
AATTTTCT White CTGT Ding ACTTGG Ding TTA
ATAGTACTTGGAGTACTAAADINGGACA
CAATCAII: CCTCCCATGCAGAATA
A^^ False GTAGGAAAAGCAATGTATGCC
CCTCCCATC! ! IAATATTACAGGGC
TGCTATTAACAAGAGAT: -MecLeuAsnGlnSerSal G 11
J ] Fixed beginning SerlleArglleGlnArl(Be,,, GlvAsnMetArclG
lnAla) [ [ 1″″ CvsG1v01%, *Gl+iF'h
eF'he1hiro = g PheAsnSerThrTrpSer1”
11eThrl-euProcSosArg
Go to 1 L゛! sAlaMetT'-! r^1aF
'to roF ThrG1vLe+stop euLeu
Dinghr?口1eAsnCvsThrAr! IF'roA
sr+Asr+Asr+ThrAr! ILys31vF
'roG1yArlA1aF'hesalThrrle
GlvLqslle(isCysAsn11eEier
ArclA1aLvsTrp^sr+AsnThr-u
sLeuArlG1uG1nPheG1%: IAsn white sr+LvsThr11e31vG1yAspProG
1u11jsalThr)IisSerPhe^S ``1
ryrCysAsnSerThrG1nLeuPheA
snSerThrTrP'hrLI:l5G1vSer
AsnAsnThrG1uG1+5erAspThr:
1eLysG1n11e11eAsnMetTrPG1
r+01uualG1v"rolleSerGII:1
01rt11eArcIC+gsSerSer＾5n1
1e4rslAspG1yG1vASnSer^Sn^
5nG1uSerGlu11ePtqeAr111F'
roG1yGlvGluAsPMet^ITyrLys
'JalSalLvslleGl:uProiA rc
4sal(jalG1nAr=G1uLL(s^rl^
:F'heLeuGlvA1aA1aG1vSerTh
rMtG1r+A1aArc(GlnLeuLeuSe
rGlvI:A1=11eG1uA1aGlnG1n)
lisLeuLcL (', lJ G 1 r+^1°
aArc! 11eLeuA1aalG)G1y11e
TrPG1+yCysSerGlyLsLtA1:3S
erTrpSer^snL+gsse rLeuGkoG
1uTrpAspArc1G1uI 1eAsnAsn
Tsr=AsPAsnTrPArtSerGluLeu
TvrLys=uGlvsalAlaProThrLv
sA1aLysArc! L2alG1y11eG1y
A1aLeuF'heLeuG1v=tG1vA1aA
1aSerMetThrLe+JThrsallesa
lG1nG1nGlnAsnAsr+LeuLeuAr
cl=uG1nLeuThrValTrpG1+11e
LysGlnLuArc1dvrLeuLvsAspG
1nGlnLet stopelJ:ulleCvsThrTh
rAlasalProTrPAsn'G1nl1eT
rPAsn^snMetThrTrPMetrThr Mee 1,7alTrpLysG1uA1aThrT
z ASP DinghrGlusualHisAs
nL, Iyo ^sr+F'roGlnGLusalL椰) ^S n A S p M e tsal
G1uG1nM/// >LeuLysF'rocysalLy
sL″7″L=G1n1-vsGluTvrA1a
F'heP= l'hrThrSerTv
rThrLe+JTa F'roLys1
w1.1lSerF'heG1uP
Aj? 'ualserThrSalG1r+
cysTLsuG1'' white e, nG1yserLeu^
hrThrLeuPheCvsAlaSerAspAl
aLysA1aTl:IralTrPAlaThrHi
sAlacysalF'roThrAspF'r.

elJζ-JalAsnL/alThrGluAsnP
heAsnMetTry-Lyset)isG1uA
sp41elleSerLe+JTrPAsPG1nS
ereuThrF'roLeuCysualSerLe
uLvsCys dinghr^S? i] rAsnserser
SerG1yAr1MetIleMetG1uS5e
rF'heAsn 工1eSerThrSer11eAr
clG1yLys81heTIJrLysLeuAs
prlerleF'rorle^5P^5nAsr-h
rsercysAsnTnrSersa111eThr
G1r+A1aCqsro11eF'ro11eHis
TyrCvsA1aF'roA1aG1yf'hesn
LysThrF'heAsnG1yThrGII:IP
roCI:l5Thr^5nhrl・(isG1y11
eArlF'rosalserThrG1r+L
pu1aG1uGIIJG1+”allalleA
rlserAlaAsnPheTiwrAqPAsnA
laLvsThrller: C°=t c, T ”t
lr A r n F' r o A (E, r+
A s n A 71 n T 'rG1yArcfA
1aF'hesalTi1r11eGkoAc,n工1e
SerArlA1aLrsTrpAc^r: 101uG
1r+F'heG1vAsnAsnLsA s P F
'ro G 11J IleSalThrHi
sse^5nSerThrG1nLeuF'heAsr
HeartεGlySerAsnAsnThrGluG1ySe
G1r+l1e11eAsr+MetTrpG1nGI
SerG1qG1n11eArlCysSerSeGl
y=・Gly8゛nSerAsnAsnG1uSeI
A111-', -〇+5-A=, r+TrpAr≦5e
rG1uhiε[esalGlnLsuAsnGlnse
rsalGlu11eAsn]rArc! LvsSer
11eArjIlsG1nArc! G1vF'r.

LvLvsIleG1vAsnMetAr! Gln white 1
aHiscvs;nAsnThrLeuLysGlr+
l1eAsPSerLysLeutsThr11eIl
eF'heLysG1n! EerSerG1yG1v:
rF'heAsnCvsGlvG1uG1uF'heF
'heTyrCvs:rThrTrpF'heAsnS
erThrTrPSerThrLvs? rASPThr
11eThrLeuF'roCvsArc111eLy
s! LilialG1vL%: 1sA1aMetTyr^1
aF'roF'roIle; rAsnIleThrGl
qLeuLeuLeuThrArlAsp; rG1u1
1eF'heArlF'roGlyGlyGlv^SP
Met；uT+rLvsT+yrLysalsualL
yslleGluF”r.

CTGTTTAAATGGCAGTCTGGCAG white AG
l'MACAGACAATGCTAAAACCATAA
TAGT〆T(3TACAAGACCCAAACAACA
ATACAAG? GGGAGAGCATTTGTTAC
AATAGGAAAI^ACATTAGTAGAGCA
AAATGGAATAA(AG80AACAATTTG
G^A8TA^Ding white^^AC〆G A C C C^G
AAATTGT^ACGCACAGTTT”.^^TT
CA＾CACA＾CTGTTTA＾TSelf GT White C “GG
GTCAAATAACACTGAWhiteGGAAGTG^(
C/1lAATTATAAAACATGTGGC86G^
White GTV＾GTGGAC＾AATTAGATGTTCA
TCAAA1? GAGGTAGTAATTAGATCT
GCCAATTTC\CAGCTGAACCAATCT
GTAGAAAATTAAT4AAAAGTATCCGT
AT'CCAGAG^GG^CC^? ATAGGGAAA
TATG^GACAAGCACATTGT:ACTTT
AAAACAGATAGATAGCAAAATTA? A.T.
AATCTTTAAGCAGTCCTCAGGAGGG
rAATTGTGGAGGGGG ownATTTTCTAC
TGTrTGGTTTTAATAGTACTTGGAGT
ACTAAA:^CAATCACCCTCCCATGC
AGAATAAA? GGAAAAGCAATGTAT
GCCCCTCCCCATCrATTACAGGGCTG
CTATTAACAAGAGAT

[Brief explanation of the drawing]

FIG. 1 - This is a flow diagram of the construction of plasmid pREV2.2 used to construct the novel protein-encoding vector. FIG. 2 - This is a diagram of plasmid piEV2.2 showing multiple cloning locations. FIG. 3 - This is a diagram of the HTLV-III envelope gene and the novel recombinant protein obtained therefrom. Figure 4 - Non-HTLV-I at the N-terminus from PBI
Diagram showing removal of II sequences. Figure 5 - Non-HTLV at the (7) C-terminus from Pet
- Diagram showing removal of m sequence. Applicant Repligen Corporation Agent Yatabe Sasai (1 other person)

Claims (1)

[Scope of Claims] 1. Recombinant HT of HTLV-III protein selected from the group consisting of R10, PB1, 590 and KH1 for treating humans in need of stimulation of lymphocyte proliferation response.
A human lymphocyte proliferation response stimulator containing an LV-III envelope protein portion. 2. The stimulant according to claim 1, wherein the recombinant HTLV-III envelope protein fragment is the HTLV-III protein portion of R10. 3. The stimulant according to claim 1, wherein the recombinant HTLV-III envelope protein fragment is the HTLV-III protein portion of PB1. 4. The stimulant according to claim 1, wherein the recombinant HTLV-III envelope protein fragment is a HTLV-III protein portion of 590. 5. The stimulant according to claim 1, wherein the recombinant HTLV-III envelope protein fragment is the HTLV-III protein portion of KH1.