JPS61233700A - Molecularly cloned aids related polypeptide - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to immunological products derived by molecular cloning, as well as methods for their use. For more information, see Acquired Immune Deficiency Syndrome (AIDS, AIDS)
The present invention relates to immunological polypeptides that are useful as diagnostic products (diagnostic agents) for the detection of viral pathogens, as well as vaccines used for vaccination against the syndrome. The present invention also relates to polypeptides of AIDS retrotranscriptase and methods of use thereof.

BACKGROUND OF THE INVENTION Traditionally, analytical studies of immune responses to various viral infectious agents have been conducted to identify such pathogens as (a) diagnostic agents for detecting immune responses against such pathogens;
)) Resistance to infection by such pathogens (tolerance)
Viral polypeptides have been limited by the fact that it has often been difficult to culture them in sufficient quantities to isolate them for use as vaccines.

Developments in molecular cloning have enabled the production of virtually unlimited amounts of gene products derived from pathogenic substances in non-pathogenic form.
Some of these limitations have been overcome by the availability of expressive means.

Influenza virus (1), hand, foot and mouth disease virus (2)
), hepatitis virus (3), small water saturated stomatitis virus (4)
), and antigens derived from rabies virus (5) are derived from Escherichia coli (
E.

However, several laboratories have reported that hepatitis B surface antigens expressed in prokaryotes do not react immunologically with antisera against naturally occurring antigens. A report has also been published (6).

Acquired immunodeficiency syndrome (hereinafter referred to as AIDS) has a significant impact on cell-mediated immunity.
It is a devastating disease of the adult immune system. This disease has a T4 helper/inducer phenotype defined by the monoclonal antibody 0KT4 (7).
-It is characterized by severe lymphatic deficiency, which appears to be due to loss of IJ lymphocytes. Other clinical findings include primarily the occasional Pneumocystis carnii
infections such as pneumonia, as well as Kaboji's sarcoma (Kapo
(7)j Pre-AIDS, a syndrome that often precedes the onset of AIDS, is characterized by chronic systemic lymph node disease. Approximately 1096 patients with PLAYAIDS have AIDS
It has been reported that this can occur (8).

The main groups at risk of developing AIDS are homosexuals and homosexual men, users of intravenous drugs, people receiving blood products (primarily hemophiliacs treated with factor II), and This includes people who have received blood transfusions (7). Epidemiological studies of this syndrome have shown that AIDS is caused by infectious agents transmitted through close contact or contact with blood or blood products (7)
.

Recent research reports include the above-mentioned HTI-V
-H-associated human retroviruses may be AIDS virus pathogens (8°9.1
0). Recently, several laboratories have isolated and propagated retroviruses associated with AIDS infection. Gallo (11) named a virus isolated from an AIDS patient, HTi, V-J, and propagated it in a permissive T-cell line (12). Similarly,
Pasteur Institute
, Paris)
Nier) discovered a virus named LAV (lymphadenopathy-associated virus) in the bodies of AIDS patients. There are reports that this LAV and HTI-V-[ appear to be similar or identical (13°14). More recently, a virus named ARV (AIDS-related virus) has been isolated, identified, and cloned from an AIDS patient. In this book, it will be referred to as AIDS-related retrovirus.

HTLV-III Q I Q k l) RNA
It is believed to be a retrovirus with a color filter. The virus particle contains a capsid made up of many proteins. The most important core protein is named P-24 and has a molecular weight equivalent to about 24,000 daltons. This P-24 core protein is the gag fff of the HTLV-Iff genome.
It is synthesized in vivo as part of a precursor polypeptide encoded by region i. This precursor polypeptide is
When processed within infected cells, P-24 and other viral proteins are formed. In addition, an envelope protein called 'gp-41 (molecular weight, 41,000 Daltons) is also a constituent of the virus particle, and is similar to f(TLV-1
It is coded in the env area of [.

Envelope protein pp- of summer iTr, v-nr
Antibodies to 41 have been detected in the serum of AIDS and pre-AIDS patients. Approximately 88% of AIDS patients and 79% of pre-AIDS patients possess detectable antibodies against f(TLV-IIr envelope protein).
16). Some researchers have reported that serological conversion to such antibodies (seroconversion) occurs at a rate of 90-100% (8).

Furthermore, in addition to antibodies directed against gl)-41 envelope protein;
-24! Antibodies against this and HTLV-■ proteins called P-55 and P-60 were detected in gp patients (16.17). Therefore, the detection of antibodies to these or other as-yet-unidentified antigens is a clear indication that one has been exposed to, or has had a productive infection with, substances associated with AIDS. ing.

Furthermore, some of these viral polypeptide sequences can also be used as vaccines to induce the production of neutralizing antibodies and confer resistance to AIDS infection. In order to facilitate the induction of high titer immune responses, undesirable viral sequences can be removed and mutations such as fusions of viral polypeptides with highly immunogenic polypeptides can be used. I need to get an array.

Therefore, the present invention aims to express in prokaryotes a viral antigen of an AIDS-related retrovirus that is non-pathogenic and can be used as a diagnostic agent for the detection of AIDS. It is something.

The present invention also aims to express in prokaryotes a diagnostic product for the detection of AIDS, which consists of a polypeptide having a mutant composition of a naturally occurring AIDS-related polypeptide sequence. be.

A further object of the present invention is to express AIDS-related polypeptides or variants thereof that can be used as vaccines against AIDS, including fusion polypeptides of AIDS-related polypeptides.

Furthermore, the present invention provides a method for analyzing AIDS RNA-dependent D for use in analysis (measurement) for identifying transcriptase inhibitors.
The purpose is to express NA polymerase (reverse transcriptase).

SUMMARY OF THE INVENTION Cloning the entire genome of an AIDS-associated retrovirus;
The sequence was determined. According to the present invention, DNA encoding an AIDS-related polypeptide has been identified and used in a recombinant prokaryotic or mammalian cell culture system to produce predetermined AIDS-related polypeptides, derivatives thereof, and the like. An amino acid sequence variant of was synthesized.

Derivatives of AIDS-associated polypeptides 1. This includes polypeptides that are unglycosylated or glycosylated with various 1 forms, as well as formylmethionyl N-terminal species (sp, ecies).

Variants of normal AIDS-associated viral polypeptides are
one or more amino acid residues of the above normal polypeptide are deleted or substituted with other residues, or one or more amino acid residues are inserted. . These variants include predetermined fragments (deletion variants) obtained from normal polypeptides, AIDS-associated viral polypeptides or fragments thereof, and second polypeptides other than AIDS-associated viral polypeptides. or fusions with fragments thereof (fusion or insertion mutants), and all of the above 1. In which one amino residue is substituted.

The present invention provides a DN encoding a polypeptide of the present invention.
A, as well as vectors into which such NAs have been operably inserted, as well as mammalian or prokaryotic cell cultures transformed thereby.

The predetermined polypeptide sequence of the AIDS-associated retrovirus produced in the present invention essentially contains (substantially) other naturally produced (existing) AIDS-related polypeptides. do not have. These polypeptides contain at least one antigenic determinant that can specifically bind a complementary antibody. Specific polypeptide sequences described herein include P-24, P-15,
E', reverse transcriptase, 6L, and envelope (env) polypeptides. In a preferred embodiment of the invention, a fragment of a predetermined polypeptide sequence of an AIDS-associated retrovirus and/or a fusion thereof, comprising at least an antigenic determinant capable of specifically binding with a complementary antibody. You can get one with one. 1 of this aspect
The C-terminal part of P-24 and the entire P-15 sequence were taken as seeds. This truncated P-24, expressed in E. coli, shows unexpected reactivity with antibodies against AIDS-associated retroviruses. Particularly preferred fragments, as well as E' polypeptide fragments, are fragments of the env protein gp120 that are capable of binding to normal host cell receptors in competition with intact virus.

The fusion polypeptide comprises (a) a predetermined polypeptide sequence of an AIDS-associated retrovirus, or a fragment thereof, having at least one antigenic determinant capable of specifically binding a complementary antibody; and (b) It contains antibodies that are normally present in a biologically collected sample for analyzing the presence of antibodies against AIDS-associated retroviruses and a second polypeptide sequence that does not react immunologically. If the fusion polypeptide is to be used as a component of a vaccine, the second polypeptide sequence may also include:
Such vaccines must not elicit (induce) antibodies that can cross-react with naturally occurring polypeptides within the target population. In this case, polypeptides obtained from lower eukaryotes other than yeast are useful.

In one type of fusion polypeptide, the DNA sequence encoding the polypeptide sequence of human growth hormone (HGr-T) is combined with HTLV-m(f)p-24 and P-
15 polypeptide sequences are located 5' to the T)NA sequence. It is expressed and processed in E. coli as an HGH-p24 fusion polypeptide that specifically binds complementary antibodies. Similarly, E'
polypeptides and enzymes such as gp41 and gp120.
Fusions of v polypeptides with synthetic or prokaryotic viral polypeptides are also obtained. These results demonstrate that viral antigens associated with AIDS can be expressed as fusion polypeptides to detect antibodies naturally produced by AIDS-infected individuals.

The polypeptides of the invention can be formulated into therapeutically effective dosage forms with pharmaceutically acceptable excipients and administered to AIDS-susceptible animals to generate antibodies against such polypeptide sequences. It can also lead to the production of

One purpose was to use RN derived from AIDS-associated retroviruses.
A polypeptide comprising or consisting of a predetermined polypeptide sequence of an A-dependent DNA polymerase (reverse transcriptase) to identify compounds that inhibit such AIDS-associated reverse transcriptase. Measurement method for (
The objective is to clone and express the protein for use in a quantitative assay. Such compounds can be administered as drugs to block infection by AIDS-associated retroviruses or to inhibit dissemination of such retroviruses within the body of an infected person.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows the amino acid sequence of the amino-terminal polypeptide of the P-24 core protein obtained from the live HTLV-11 [retrovirus] and the corresponding sequence deduced therefrom. Shows the DNA sequence.

Figures 2a-2j depict the f-IT L V -N genomic DNA sequence, deduced polypeptide sequence, and partial restriction map. The terminal repeat sequence ("k") and specific regions (U5 at the 5' end, and U3 at the 3' end) are indicated. gag region, reverse transcriptase (“pol”), envelope (“env”)
”) as well as the putative positions assigned to the E' and P' proteins. sd and sa represent the splice donor and splice acceptor sites, respectively. The 13 overlapping amino acid sequences are ,
A putative mRNA splicing event is represented. The bases listed alternatively indicate the differences found between the clones and represent the variations observed between strains of the virus population.

The 542k diagram is a restriction enzyme map of proviral DNA and a group of cDNA clones that overlap with this.

FIG. 21 is a diagram showing the putative mRNA splicing activity of the virus, as well as the obtained mesengers and proteins encoded by them.

FIG. 3 is a schematic diagram showing the assembly scheme used to subclon p7.11.

Figure 4 is a schematic diagram of the assembly of the P-24 expression vector and the truncated P-24 expression vector. Figure 5 is the P-24 expression vector.
FIG. 4 is a schematic diagram of the construction of expression vectors for a 4-compos ite polypeptide as well as for a truncated P-24 hybrid polypeptide.

Figure 6 shows I) DE-24, PDE24△HD3, pH
GHp -24△B and p HGHp -24△HD
This is an image obtained by subjecting the polypeptide expressed by No. 3 to Western blotting. Rabbit anti-HTLV-■ antibody was used to process the plots.

FIG. 7 is an image obtained by Western blotting of the polypeptide expressed by pDE-24. Human serum obtained from AIDS patients was used to process the plots.

Sections 8a, 9a and 1. Oa diagram shows plasmid ptr
pLE, gp41, ptrpr, E, E' and pSV
E,E'r) It is a schematic diagram showing the structure of fIFR.

Each of these plasmids r-ITLV-111
env protein and E' protein fusions in bacteria, and the unfused E' protein in the CI-10 cell line.

Figures 81), 91) and 101) are pt.
Protein fusions encoded by rpLE, gp41, ptrpLE, E' and pSV
E.

E' r) Unfused E' encoded by HFR
FIG. 2 is a reproduction of a polyacrylamide gel electrophoresis pattern showing protein synthesis.

+1112 Figure shows e in the culture of recombinant eukaryotic cells.
IIT L used to prepare nV protein
V-1'[This is a diagram showing the region of the genome.

FIG. 11b is a schematic diagram showing the expression plasmid used to secrete I(TLV-m envelope protein) into cultures of mammalian cell culture transformants.

Applicants have demonstrated that viral proteins from AIDS-associated retroviruses can be expressed in host cells, either directly or as mutant polypeptides, and that such recombinant polypeptides can be expressed directly or as mutant polypeptides in host cells. It has been demonstrated that it can specifically bind to antibodies. Such mutant polypeptides include fusion polypeptides of a viral polypeptide and one or more second polypeptides, as well as polypeptides containing deletions, insertions, or substitutions in the viral polypeptide. and derivatives of the polypeptides. Furthermore, any polypeptide containing a fragment of a predetermined polypeptide sequence of an AIDS-associated retrovirus, whether expressed directly or with certain mutations, It was found that it reacts with antibodies against retroviruses. These results indicate that such recombinant polypeptides can be used as diagnostic agents to detect AIDS in individuals, donated blood, and facial fluid products.

Furthermore, such polypeptides can be used as immunogens to induce the production of neutralizing antibodies that confer resistance to infection by AIDS-associated retroviruses.

Furthermore, infection with AIDS-related retroviruses is
Reverse transcriptase of AIDS-associated retroviruses can be used to identify compounds that can stop [m] or prevent dissemination of such retroviruses in infected individuals.

The fusion polypeptide of the invention consists of an AIDS-related polypeptide sequence and a second polypeptide sequence. This second polypeptide sequence is used to: ■) promote secretion of the fusion polypeptide from the bacterial host into its extracellular environment or into the Durham-negative periplasm; or 3) to provide a polypeptide sequence that can be used for purification of the fusion polypeptide (e.g., specific for I(GH)). It is used when purifying the I-IGH-AIDS fusion polypeptide by fractionation using an immunoadsorbent.Depending on the individual application, forming the fusion polypeptide with an AIDS-related retrovirus. The second polypeptide sequence used to induce AIDS may be of prokaryotic or eukaryotic origin and may be located at either the amino terminus, the carboxy terminus, or both termini of the AIDS-related polypeptide sequence. Furthermore, it may be inserted inside the AIDS-related polypeptide sequence.

a second component used to promote secretion of the fusion polypeptide;
Examples of polypeptide sequences include (1) the signal sequence of the herpes simplex virus protein disclosed in pending U.S. Method Application No. 527,917 (filed August 30, 1983); (2) Signal sequence of E. coli alkaline phosphatase or E. coli enterotoxin ST [pending U.S. Method Application No. 658.342 (Oct. 1984)]
F$) and disclosed in the documents cited in the application]; In addition, signal sequences derived from higher eukaryotes (for example, the signal sequence of gamma interferon, etc.) are included.

An example of a second polypeptide sequence that facilitates intimacy with a functional membrane is the transmembrane transport sequence of herpes simplex virus [US method application rate 527.9].
No. 17 (filed on August 30, 1983)].

Many people who are at risk of AIDS infection also have antibodies against E. coli and other enteric bacteria, so a secondary antibody that does not cause a false immunopositive reaction with these antibodies may be used.
polypeptides must be selected. Therefore, the use of polypeptide sequences obtained from enterobacteriaceae as the second polypeptide is advantageous since such sequences would otherwise be removed during processing.
Sequences that inhibit reactivity with biological samples for analysis of the presence of antibodies produced in response to infection with AIDS-related viruses [e.g., by recombinant methods, The epitope of the bacterial protein has been modified so that it can no longer cross-react (see 1.E fusions below).

When the fusion proteins of the present invention are used as a vaccine against AIDS infection, the second polypeptide sequence must be modified so as not to generate antibodies against the naturally occurring polypeptide in the subject to be vaccinated with such a vaccine. must be selected. For example, in the case of a vaccine for human use, the second polypeptide sequence is preferably not HG11. However, such vaccines may also contain prokaryotic or, preferably, eukaryotic polypeptide sequences other than yeast and primate.

In the present invention, specifically, a certain I-ITLV-1')-
Cloning and expression of encoded polypeptides is disclosed. However, the present invention is applicable to other HT L
It also encompasses the cloning and expression of V-ffr polypeptides. HTLV-Iff polypeptides containing antigenic determinants for antibodies to AIDS and AIDS patents include g
l) -160, gp-1,20, gp-65, gp-
41, p-60/p55. The gp-120 polypeptide appears to be a precursor polypeptide for gp-65 and g,p-41. These specific HT L
The V-]'l'J polypeptide is exemplary and is not intended to limit the scope of the invention.

Furthermore, the present invention provides a library of products, each having a different antigenic determinant, which can be used to determine which antigenic determinant(s) are optimal for the detection of AIDS or AIDS. This also includes generating. Such a library may be used, for example, to determine which antigenic determinants are immunologically reactive to serum obtained from healthy individuals who are serologically positive for AIDS. I can do it. Test results for such serum are positive, but blood aI obtained from AIDS patients
Antigenic determinants that show negative results in this test are the first candidates for vaccines to induce the production of neutralizing antibodies. Additionally, diagnostic products (diagnostic agents) containing such antigenic determinants may be used to identify people who have neutralizing antibodies but who are unlikely to progress to severe symptoms associated with AIDS. be able to.

It should be understood that the present invention is based on research related to HTLV-1 [), and that HTLV-[ and LAV or ARV are similar or identical. As such, polypeptide products derived from these retroviruses are also included within the scope of the present invention. Accordingly, the term AIDS-associated retrovirus refers to HTLV-■, LAV, ARV and/or other retroviruses capable of causing AIDS or pre-AIDS.

As used herein, the polypeptide sequence of an AIDS-associated retrovirus is either a full-length naturally occurring polypeptide sequence or a predetermined sequence derived from genome sequencing.

A naturally occurring (occurring) polypeptide sequence is
polypeptides formed within cells infected by viruses;
or a polypeptide found in the culture medium of such cells.

Variant polypeptide sequences of AIDS-associated retroviruses include: (1) fusions of a viral polypeptide or a fragment thereof with a second polypeptide (fusions at the N- and C-termini, as well as insertional fusions); include) ;
(2) Fragments of polypeptide sequences of AIDS-associated retroviruses generated by deletions in the N-terminus, C-terminus, or intermediate region of viral polypeptides: (3) Polypeptides of AIDS-associated retroviruses and (4) labeled by well-known methods or as disclosed, for example, in U.S. Method No. 3,720,760 (see bibliography). Included are derivatives such as labeled or conjugated viral polypeptide sequences that are attached to a solid phase according to the method.

Ru.

1 "Second polypeptide sequence" refers to a sequence that is fused with an AIDS-associated retrovirus polypeptide sequence or a fragment thereof to form a fusion polypeptide sequence of the invention. These second polypeptides are nuclear organisms, non-
Any sequence or partial sequence of a protein derived from the AIDS virus or a prokaryote that promotes secretion of the fusion polypeptide, but facilitates the intimacy of the fusion polypeptide with the surface membrane of the expression host. , or to purify fusion polypeptides. When used as a vaccine, the second polypeptide of the fusion polypeptide under normal conditions
Sequences that are incapable of inducing (raising) antibodies that can cross-react with naturally occurring polypeptides in the subject for whom such a vaccine is intended.

"Complementary antibody" refers to an antibody raised against a corresponding naturally occurring viral epitope, or an AIDS-encoded epitope that is not packaged within virians. The DNA sequence of the AIDS-associated retrovirus encodes the polypeptide sequence and variant polypeptide sequences of the present invention as described in "U".

“Biologically derived sample” includes any biological fluid or tissue sample obtained from a human or animal subject that has been exposed to an AIDS-associated retrovirus or Included are samples that are subjected to analysis to detect the presence of complementary antibodies produced in response to infection with the virus. Such samples are generally blood, urine, semen, and saliva, but include any biological material in which such complementary antibodies or AIDS-associated retroviruses are found.

Prokaryotes are preferred for cloning and expressing DNA sequences to produce the diagnostic products (diagnostic agents) and vaccines of the invention. For example, 1.2 strains of E. coli29
4 (ATCC 3]446) is particularly useful. In addition, E. coli B and E. coli X1776 (ATCC Cup 3
15-37), as well as E. coli C600 and C600h
f 1, Escherichia coli W3110 (F-1λ-1 prototrophic strain,
E. coli strains such as ATCCA27325), Bacillus subtilis (B
strains of the genus Bacillus, such as Salmonella typhimurium (Salmonella typhimurium).
monellatyphimurium) or Serratia marcesans (5erratia marce)
Other strains of microorganisms can also be used, including other Enterobacteriaceae, such as Pseudomonas sans, as well as strains of various Pseudomonas species. When expressed in prokaryotes, the polypeptides of the invention typically contain N
- Contains methionine or formylmethionine at the terminal, but is not glycosylated. Of course, the above is intended to be illustrative and not limiting.

Generally, plasmid vectors containing replication and control sequences derived from a species compatible with the host cell are used in connection with such a host. usually,
The vector has a sequence encoding a protein that can confer phenotypic selectivity to transformed cells, and a replication site.

For example, E. coli is commonly transformed with pBR322, a plasmid obtained from E. coli species (18).
. Plasmid pBR322 contains genes for ampicillin and tetracycline resistance, which provide a means by which transformed cells can be easily identified.

This pBR322 plasmid, or microbial plasmid, also contains, or must be modified to contain, a promoter that can be used by the microorganism to express its own proteins.

The most commonly used promoters for recombinant DNA assembly include the β-lactamase (penicillinase) and lactose promoter systems (19-21), and the tryptophan (trp) promoter system (22,23).
) is included. Although these are the most commonly used promoters, other microbial promoters have also been discovered and used, and their nucleotide sequences have been published in detail, so that one skilled in the art will be able to identify them and function with plasmid vectors. can be operably ligated (24). In the example already disclosed,
The trp promoter (22, 23,) has been used to express the diagnostic products and vaccines of the invention.

In addition to prokaryotic cells, eukaryotic cells can also be used to express AIDS-associated viral polypeptides, including reverse transcriptase of AIDS-associated retroviruses, among others. Among eukaryotic microorganisms, Satucharomyces cerevisi x (
Saccharomyces cere-visia
e) or regular baker's yeast is most commonly used, but many other strains can also commonly be used. For expression in Saccharomyces, eg, plasmid YRp7 (25-27) is typically used. This plasmid already contains the trpl gene, which is either ATCC&, 44076 or PEP4-
1 (28), it serves as a selection marker for selecting yeast mutants that lack the ability to grow in tryptophan. Since this yeast host cell genome is characterized by a trpl defect, the fact that it grows in the absence of tryptophan provides an effective environment in which transformants can be detected.

Suitable promoter sequences for yeast vectors include 3-phosphoglycerate kinase (29) or enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6 - phosphate inmerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate inmerase, phosphoglucose inmerase,
and other glycolytic enzymes such as glucokinase (30,
3], ) includes non-promoted items. To construct a suitable expression plasmid, the termination sequences associated with these genes are ligated into the expression vector 3' to the desired sequences to be expressed, such that the mRNA terminus is polyadenylated. Make it.

In addition, promoters that have the advantage that transcription is controlled by growth conditions include alcohol dehydrogenase 2, isocytochrome C1 acid phosphatase, degrading enzymes related to nitrogen metabolism, the glyceraldehyde-3-phosphate dehydrogenase, and the promoter regions of enzymes involved in the utilization of maltose and galactose. All plasmid vectors containing yeast-compatible promoters, origins of replication, and termination sequences are eligible.

Multinucleated cell cultures can also be used to express AIDS-associated retroviral proteins.

VERO and HELA cells, Chinese hamster ovary (CHO) cell lines, and w438, BHK
Of particular interest are mammalian or mammalian cells such as the ICO57 and MDCK cell lines. Expression vectors for such cells typically include an origin of replication and a promoter to control expression of the coding DNA of the AIDS-associated retroviral polypeptide, and, if necessary, a mammalian selectable marker, an RNA splice site, , along with a polyadenylation site and a transcription termination sequence. Vectors capable of transforming mammalian host cells to express AIDS-related polypeptides include r)f(
A selection marker, such as the gene encoding FR (dehydrofolate reductase), is introduced into host cells by known methods, and the transformants are then exposed to increasing concentrations of a selection agent (e.g., methotrexate). Exposure and amplification (see US Method No. 4,399,216) is preferred.

Transcriptional and translational control functions for use in mammalian cells are routinely obtained from viral sources. Commonly used promoters are obtained, for example, from polyoma, simian virus 40 (SV40) and especially adenovirus 2. The early and late promoters of the 5V4Q virus are useful, as are the major late promoters of adenoviruses described above. Additionally, promoters or control sequences normally associated with the desired gene sequence can also be used, and are often preferred, provided such control sequences are compatible with the host cell system.

The origin of replication is adenovirus or other viral origin (
For example, polyoma, 5v40, VSV.

BIPV) or, if the vector is integrated into the chromosome of the host cell, by the chromosomal complex IS+ of the host cell. .

If the vector of the present invention contains a DNA sequence encoding both an AIDS-related polypeptide and a gene involved in co-transformation, selection and amplification, such as the DHF R enzyme, the DI used - (It is appropriate to select a host depending on the type of FR protein. Wild type D
When using HFR proteins, it is preferred to select DHFR-deficient host cells, thereby selecting for transfections with a high Fi = 47) in selection medium lacking hypoxanthine, glycine, and thymidine. D I-I F R as a marker for
A cryptographic sequence can be used.

On the other hand, DI with low binding affinity for MTX (when using FR protein as a control sequence, D
There is no need to use HFR resistant cells. Since the mutant D HFR is resistant to methotrexate, MTX-containing media can be used as a means of selection if the host cells themselves are sensitive to methotrexate.

Alternatively, the wild-type DI (FR gene) can be used as a marker for amplification in host cells that are not DHFR-deficient, provided a second drug selectable marker is used, such as neomycin resistance.

In the Examples described below, CHO cells are used as host cells, and an expression vector encoding reverse transcriptase of an AIDS-related retrovirus is used.

As detailed below, the diagnostic products of the invention can be used in similar immunoassays in place of their counterparts derived from live pathogens. From this point of view, commercially available diagnostic test kits include
The diagnostic products described above can be included together with a variety of other immunological products, provided that at least one of these is labeled for the detection of a complementary antibody or antigen. ). This system is useful for the molecular cloning and expression of specialized proteins of HTLV-1 that have sufficient antigenic determinants to specifically bind complementary antibodies, i.e., antibodies to HTLV-1'. I have explained. Specific methods used for "cloning and expressing exemplary polypeptides" are detailed in the Examples below.

Many methods are known for quantifying unknown amounts of antigens or antibodies contained in biological fluids such as serum, urine, or saliva, or in skin specimens. Basically, the present invention employs such known methods, except that they are replaced by certain molecularly cloned diagnostic reagents of the type described above.

Therefore, for details of the method, reference will be made to conventional immunology textbooks, and the method itself will be described in a general manner. Those skilled in the art will be familiar with how to utilize the novel diagnostic products of the present invention in conventional immunological methods.

For ease of description, the general term "diagnostic product" or "diagnostic agent" is used herein to refer to the antigenically functional products of the invention.

1. The term "diagnostic product" refers to a predetermined diagnostic product of an AIDS-associated retrovirus that has one or more antigenic determinants capable of specifically binding to complementary antibodies raised by the AIDS-associated retrovirus. Define it as a polypeptide sequence. This diagnostic product is formed within a recombinant host cell capable of producing it. The polypeptide sequence may be functionally linked to the surface membrane of the recombinant cell, or may be recovered and used free of the cell membrane of the host cell. Furthermore, this antigenic polypeptide sequence may be fused to a second polypeptide sequence.

Diagnostic methods used to measure AIDS-associated retroviruses, their constituent polypeptides, and complementary antibodies are conventional. These include the competitive method, the Sandermanch method, and the steric hindrance method. In the first two methods, phase separation is an essential step in carrying out these methods, whereas the steric hindrance method is carried out in a single reaction mixture system. Although methods for measuring retroviruses or their polypeptides and methods for measuring substances that bind to retroviruses or viral polypeptides may be preferred depending on the size of the substance to be measured, these The method is essentially the same. Therefore, in this specification, a test substance is referred to as an analyte (analyte), whether it is an antigen or an antibody, and proteins that bind to this analyte are referred to as antibodies, cell surface We will refer to it as a binding partner, whether it is a receptor or an antigen.

All assays involving AIDS-associated retroviruses, their polypeptides, complementary antibodies, or cell surface receptors use one or more of the following reagents: labeled analyte analogs. immobilized analyte analogs, labeled binding partners, immobilized binding partners, and steric conjugates.

Labeled reagents are also referred to as "tracers."

The label used may have any detectable function so long as it does not interfere with the binding of the analyte to its binding partner. Labels used in immunoassays include enzymes such as horseradish peroxidase, 14C and 1
Many are known, including radioactive isotopes such as 311, rare earth chelates, fluorescent compounds such as fluorescein, and spin labels. These labels and proteins or polypeptides can be covalently bonded in a conventional manner. Such binding methods are suitable for use with AIDS-associated retroviruses, viral polypeptides, complementary antibodies, and retroviral receptors, all proteinaceous substances.

Certain assay methods require immobilization of reagents.

Immobilization allows the binding partner to be separated from any analyte remaining free in solution. This means that before starting the measurement of binding partners or analyte analogues, they must be adsorbed onto a water-insoluble matrix or surface [Bennig (Benig, B.
et.
This can be accomplished subsequently by any method, such as by insolubilizing these partners or analogs by subjecting them to immunoprecipitation, for example.

Steric conjugates are used in steric hindrance methods for homogeneous assays. These conjugates covalently link a low molecular weight hapten to a small analyte, allowing antibodies directed against the hapten to bind to the conjugate simultaneously with the -1-1 anti-analyte. It is synthesized in a way that is impossible. In this assay, the anti-hapten and the conjugate bind to the analyte present in the test sample and the anti-analyte, and as a result, when the hapten is a fluorophore, will cause a change in fluorescence.

Other assay methods, also known as competitive methods or the Sanderch method, are also well-established and widely used in the commercial diagnostic industry.

Competition assays rely on the ability of a labeled analogue (“tracer”) and an analyte in a test sample to compete for a limited number of binding sites present on their common binding partner. This is the way to base it. Typically, the binding partner is insolubilized before or after competition, and the tracer and analyte bound to the binding partner are then separated from the unbound tracer and analyte.

This separation is carried out by decanting (if the binding partners have been previously insolubilized) or by centrifugation (if the binding partners are precipitated after a competitive reaction). The amount of test sample analyte is inversely proportional to the amount of bound tracer determined from the measured amount of marker substance. Prepare a dose-response curve for a known amount of analyte and compare this with test results to quantitatively determine the amount of AIDS-associated retrovirus, viral polypeptide, or complementary antibody in the test sample . In these heterologous measurement methods, a system that uses an enzyme as a detectable marker is referred to as an ELiSA system.

Another type of competitive measurement method does not require phase separation and is a homogeneous measurement method.

In this assay, a conjugate of an enzyme and an analyte is prepared such that when the anti-analyte and analyte bind, the enzyme activity is altered by the presence of the anti-analyte. In this case, a polypeptide of an AIDS-associated retrovirus or an immunologically active fragment thereof,
Trifunctional organic bridge structures are used to conjugate enzymes such as peroxidase. The conjugate product is used with a complementary antibody and is selected on the basis that binding to this complementary antibody impairs or enhances enzyme activity.
It is widely used under the name MIT.

Sandwich assays are useful for measuring polypeptides (i.e., high molecular weight molecules) such as AIDS-associated retroviruses, complementary antibodies, or retroviral cell surface receptors.
Particularly useful. In the continuous Sanderutsch method,
Using an immobilized binding partner, a test sample analyte is adsorbed onto it, the test sample is removed by washing, the labeled binding partner is adsorbed onto the bound analyte, and then the bound substance is absorbed. and the remaining tracer.

The amount of bound tracer is directly proportional to the amount of test sample analyte. The “simultaneous” sandwich method does not separate the sample before adding a labeled binding partner. This is just an example of a method for measuring viral cell surface receptors. Methods developed today or in the future for the measurement of these analytes are also included within the scope of the present invention.

To simplify the description of the examples, well-known methods that frequently appear in recombinant assembly are abbreviated to short phrases.

Plasmids are designated by beginning with a lowercase p and/or followed by an uppercase letter and/or number. The starting plasmids or DNA sources of the present invention are commercially available, or are generally available on a limited basis, or can be assembled from available plasmids or polynucleotides by published methods. Additionally, other equivalent plasmids are known to those skilled in the art;
for the preprotein and its control sequences,
Other equivalent plasmids will be obvious to those of ordinary skill in the art, or simply serve as replication vehicles for those elements in the intermediate assembly.

"Digestion" of DNA refers to catalytically cutting DNA with an enzyme that acts only on certain positions of the DNA. Such enzymes are called restriction enzymes, and sites specific to each enzyme are called restriction sites.

The various restriction enzymes used in the present invention are commercially available, and reaction conditions, cofactors, and other requirements were used according to the instructions of the enzyme supplier. Restriction enzymes are generally listed in abbreviations consisting of a capital letter indicating the microorganism from which each restriction enzyme was originally obtained, followed by other letters and usually a number. Generally, about 1 μg of plasmid or DNA fragment is used with about 1 unit of enzyme in about 20 μl of buffer. Appropriate buffers and substrate amounts for particular restriction enzymes are specified by the manufacturer. Incubation times are typically about 1 hour at 37°C, but may vary according to the supplier's instructions. After incubation, the proteins are extracted with phenol and chloroform, and the digested nucleic acids are recovered from the aqueous fraction by ethanol precipitation. After digestion with restriction enzymes, the 5' end phosphate is occasionally hydrolyzed with bacterial alkaline phosphatase and ligation (below)! In this case, the two restricted cleavage ends of the DNA fragment are
By ``circulating'' or forming a closed loop, it is difficult to insert other DNA fragments into the restriction site. Unless specified otherwise,
Digestion of the plasmid shall not involve dephosphorylation of the 5' end. The method and reagents for dephosphorylation follow conventional methods (32).

``Recovery'' or ``isolation'' of a specific DNA fragment obtained by digestion with a restriction enzyme means that the digested product is separated by polyacrylamide gel electrophoresis, and the mobility of the fragment is compared with that of a marker DNA fragment of known molecular weight. identify the desired fragment by comparing;
This means removing the portion of the gel containing the fragment and separating the DNA from the gel, usually by electroelution. This method is generally known.

1 "Western blotting method" is a method to confirm the presence of a polypeptide by reaction with a labeled complementary antibody.The polypeptide is separated by poly-lyacrylamide gel electrophoresis, and di-trocellulose. - electrophoretically transferred to. The nitrocellulose is incubated with labeled complementary antibodies, unbound antibodies are removed, and the remaining label is located.

"Transformation" refers to the introduction of DNA into an organism in such a way that the DNA can be replicated as an extrachromosomal component or as an integrated component within a chromosome. Unless otherwise specified. , the method used in the present invention is the Ca Cl 2 transformation method (33).

"Ligation" refers to the process of forming a phosphodiester bond between two double-stranded nucleic acid fragments (34). Unless otherwise specified, ligations were performed using known buffers and conditions. This is carried out using approximately equimolar amounts of 10 units of T4 DNA ligase ("ligase") per 0.5 μg of DNA to be ligated.

"Fill-in" refers to repairing sticky-ended (protruding) restriction enzyme fragments. Generally, 2-15 μg of DNA is added to 50 mM
NACI, 10mM) Lis (pH 7,5), lQmM
MgCJ' in 1mM dithiophreitol;
Incubate for 30 minutes at 24°C with 2SQmM each of the four deoxynucleoside triphosphates and 8 units of DNA polymerase Klenow fragment. The reaction was terminated by extraction with phenol and chloroform, followed by ethanol precipitation.

I) "Preparing" NA from transformants means isolating plasmid DNA from a microbial culture. Unless otherwise specified, the alkaline/SDS method was used (
34).

"Oligonucleotides" are short, single- or double-stranded polydeoxynucleotides that are synthesized by known chemical methods and then purified on polyacrylamide gels (35).

The following examples are presented specifically to more fully illustrate the essence of the invention and are not intended to limit the scope of the invention.

Example 1 This example discloses the construction of plasmids and expression of the P-24 core polypeptide of LV-III in a prokaryotic expression host.

A, Virus multiplication and viral RNA release H-1
'LV-IIIB/I (g cell (A'I'cc CRI
-854,3), Arthur CDr, Larry Ar
thur, 7 Rederik Cancer Research
Facility (Fre-Derick Cancer
Research Facility), 7 Fred'erick, Maryland (MD)]
Obtained from. Cultures were kept in suspension and 5% F
B S, 2mM Shiichi Glutamine and 100μg/!
The mixture was adapted to RPMI 1640 fully supplemented with 1 penicillin-streptomycin mixture. Inside with capacity 10,
Cells were plated at a density of 3 x 105 viable cells/me (seeding, approximately 96 hours after seeding, 1O-15 x 10
5 viable cells/ml! Cultures were harvested when they reached a density of .

After cell removal and clarification, the culture fluid was concentrated by ultrafiltration. Beckman L8-
Using M ultra 2 filter, Beckman s w -270
- in a 20-60 sucrose gradient in a 27.00 g
By treating with Orpm for 16 hours, ultra-Pif4
The concentrate was banded to purify H'l r-V-III virus particles. Collect (pool) the 30% to 40% sucrose fraction, '1' N E [I 10
mat tris(pl'-17,5), 0. IM Na
C,e, 1mM El)'1'A:] and pelleted by ultra-differential centrifugation in a Beckman type 50 rotor. Viral particles were resuspended in TNE at approximately 2000 times the concentration of the starting medium. These concentrates are 1 me
Each sample contained 2.5-3.0 wLg of protein.

Total RNA was extracted from TLV-III B/Hg cells [Bager et al.
), Biochemistry
, ↓Abori, p. 5143 (1979)].

RNA containing polyA was purified by fractionation on oligo dl cellulose [Aviv et al.

etal), Proceedings of the National Academy of Sciences (USA') (P, N,
A, S, U, S, A, )1. , □, p. 5463 (1977)], CI) was used for the synthesis of NA.

For cDNk synthesis catalyzed by reverse transcriptase, poIJ
Primers were created by pairing ARNA with oligo d'T [Capon et al., Nature, p. 304.507 (1983)].
].

Synthesis of the second DNA strand by DNA polymerase I was self-priming. The generated double-stranded DNA is s l
By treatment with nuclease, the generated loop was nicked and the -terminal nucleotide was removed.

1 (TLV-1) using this blunt-ended double-stranded DNA.
11 phage library
y) was created.

Using T-41J gauze, the above double-stranded DNA was
R-11J linker (37) synthesized using a known technique (35)
) was subjected to blunt end ligation. This R-l/cDN
A was ligated to R1-digested λG''[10 to create H'l'I,V-IIIc as disclosed (37).
A DNA library was created.

To perform in situ hybridization (32), phage library replicates were replica plated twice on nitrocellulose to which the DNA was immobilized. I-1 used to create the cDNA library
'1- L V -III infection - cell-derived poIJ
32p-labeled c prepared from A-positive RNA 1) N
In A - set of filters was hybridized. The other set of filters was hybridized with 1(-IIV-In uninfected, from the same cell line, 32P-labeled cl)NA.

Methods used for bacterial cDNA clone detection (
A large number of H-I L V-III positive clones could be identified by a differential phage hybridization method similar to 38).

cl) NA phage library-derived [
-1L V-III clone was digested with EcoRI.

This cDNA DNA was extracted from the whole human body and pBR322 (ATCC
37017) and grown in E. coli 294 (ATCC 31446).

These multiple subcloned RI inserts were sequenced by the dideoxy method (39), yielding the proviral sequences shown in Figures 2a-2d.

Amine-Terminal Sequencing Viable 1 (TLV-III) virus particles were purified as described above. Their constituent proteins were purified using Laemmel i buffer (42).
Fractionation was performed on a 2-fold polyacrylamide gel. Approximately 600 μy of each virus protein was added to lanes (la) under reducing conditions.
(41). Coat the gel (C
ooma sie) stain with blue, 24.
．． A protein band with a molecular weight equivalent to 1,000 daltons was excised from the gel.The protein was electroeluted (42) from the gel for amino-terminal sequence analysis (42).
1) Let it happen. The sequence of the first 17 amino acids, other than residue 16, is shown in FIG. The corresponding DNA sequence is shown directly below the amino acid sequence. Comparison of this DNA sequence with the sequence of the Hl LV-III genome in Figure 2 revealed one specific clone containing the DNA sequence encoding this peptide sequence, shown at p7.11 in Figure 3. was revealed. This approximately 2.2 kb (kilobase) contains the precursor gag region, and from 5' to 3' are P-12, P-15, and the second P-15 protein, P-15. About 300 extra base pairs are encoded between the 24, 3' and gag regions. The DNA sequence of this precursor gag region and the corresponding amino acid sequence are shown in Figure 2. 22 containing the R-1 site 3' from the carboxy-terminal amino acid
A partial restriction map of the P-24 core protein region consisting of 0 amino acids is shown at P-7 and P-11 in FIG.

, p 7.11 subcloning Plasmid 7.11 was digested with F, co R1. 2゜double digestion. Rsal-PstI and Ps(I-E
The coRl sequence was isolated. Plasmid tlc9(0
1) prefecture 11 and dan (o R1. p [1
isolating a large fragment containing the majority of C9;
It was hybridized with human I-ri'' and fragment I-EcoR1 fragments. RsaI and sm
aI Both blunt ends were ligated with 1-4 ligase. It was named pUcp-24. The resulting plasmid was used to transform E. coli 294.

In the assembly of pUcp-24, P-7,11
The RsaI site was used. By doing so, the first 10 N-terminal amino acids were excluded from this assembly. Assemble a synthetic oligonucleotide to insert these 10 amino acids (5).

The DNA sequence of Ko(7) 39-mer is:
Expression: The 5' end of this oligonucleotide was attached to the synthetic silica 1 (T) for the subsequent ligation.
Contains parts. Both strands were phosphorylated with polynucleotide kinase.

Plasmid p[]c-24 was digested with BlmHI and ΣII. Most of this pUC plasmid and P
A large fragment containing the 3' end of the -24 DNA sequence was isolated. Plasmid 7.11 was digested with Rsal and ΣenkoI. 5' of p-24 DNA sequence←
A fragment containing up to the Sph I site was isolated. This fragment and the synthesized 39-mer were hybridized with the cleaved-5ph I fragment obtained from pUc-24. 39-mer and Rsa
The blunt end from the I-5ph I fragment was
Ligation was performed using 41J gauze. This, p[JCp
E. coli 294 using a plasmid named 24I-
was transformed.

C0p-24 core protein and truncated P-2
FIG. 4 shows a schematic diagram of the assembly of the 4-core protein expression vector P-24 and the truncated P-24 expression vector.

The assembled plasmid pSRCex 16 (44) of pHGf(-5RC-fs 1-2)
22, and the first 69 nucleotides of HG i (and S
60 of RC. It contains a fusion of OQQMw with DNA encoding a phosphoprotein (the fusion occurs under the control of the E. coli trp promoter). It does not matter what protein the plasmid encodes; Rather, it may be a PBR322 plasmid derivative as long as it contains the trp promoter (45).

To destroy the E (oRI site) in the trp promoter, plasmid psRcex16 was partially digested with Eco
Now) while filling the fragment with Ec at the beginning of the HG I-I fragment.
Leave the oRI site. Then the generated plasmid pE
ARI 5R (lee x l 6) was digested with ShuRI to cleave the plasmid and modified with SI nuclease to make the ends blunt, creating a linker with the following structure: 5′PAA and M]πCAT 3. - (ES l-2 Therefore, assemble p-24/p below.
The first base of the -15 first codon is supplied by the vector. This plasmid was transformed into pHGH-5RC-Es
It is called l-2.

Construction of the full-length P-24 expression vector Plasmid UCP24I- was cut with μtmH11.

The cleaved fragment was treated with DNA polymerase I in the presence of Q, 5 mM nucleotide triphosphate.
to fill in the Danba HI restriction site. This fragment was then digested with coRI. P-24 and P-15 (P-24/P-15)
I) A fragment containing the NA sequence was isolated.

EcoR plasmid HGH-5RC-fs I-2
I and filled as described above. Subsequently, the cut plasmid was digested with BamHI. A fragment containing the trp promoter and ATG start codon was isolated. This fragment was combined with the tetracycline-resistance-conferring fragment obtained from digested pBR322 by Ma-Hen) II and Shikage kI digestion, and the fragment conferring P
Blunt-ended Hemu HI-E obtained from UCP241-
Hybridized with coRI fragment. The blunt ends were ligated with T-41J gauze. P24
1) Using this plasmid called E. coli 294
was transformed.

Transformants were selected by their tetracycline resistance. Expression of this plasmid is expected to produce a polypeptide consisting of full length P-24 core protein and P-15 protein.

Construction of the expression vector for the P-247 fragment Plasmid P-241)E encodes amino acids 178-179 of the P-24 core protein l)NA
Contains a HindIII restriction site located near the sequence. Additionally, P-241)E contains a Norikuni-In site in the pBR322-derived tetracytaliph stiffness gene located 3' to the P-241)NAA sequence.

Digest P-241)E with μl1ndIII, then remove amino acids 179-200 and the fragment encoding the P-15 protein, and generate a fragment containing the 5'P-241)NAA sequence under the control of the Lrp7'' promoter. Religation of p-1
) E-24al-11) A plasmid designated 3 was constructed. The truncated P-241) NAA sequence has a TAA stop codon in the pBN222 sequence 3', which encodes the first 178 amino acids of the P-24 core protein and pBR322'') NAA sequence. amino acid residues are expressed.

Example 2 In this example, 1 (T1.V −
The construction and cloning of expression vectors expressing 111 P-24 core protein sequences as fusion polypeptides is disclosed.

A, p-24 core protein and truncated P-2.
A schematic diagram of the construction of a vector for expressing a hybrid polyheptide of fragments of P-4 and P-24 is shown in FIG.

Stand 1 Lasmid UCp24. I- as above, the lesson H
It was cut with I, blunt ended and digested with EcoRI.

A fragment containing the P-24/p-15 DNA sequence was isolated.

Plasmid HGH20? -1(4,5) was digested with 4 patches ■ and Pvu□II. Ps(I site to Pvu
□ We isolated a sequence encoding up to 11 sites of 1 (GH3').
The last eight amino acid residues of the sequence and mature f (GH have been removed). These two fragments were combined into pBR32, which contains the gene conferring tetracycline resistance.
#Hybridized to the pigeon tl-E-R1 fragment derived from No. 2. After ligation of the PvuII site and the blunt-ended Babase H1 site using T-41J gauze, the resulting Blasmi coliform 294, designated A, was transformed with p I-TG Hp 24-.

Plasmid pHGIIp24 A contains pI-(Gl(
3; The mature HG i (does not encode amino acid residues, encodes such residues) NA sequence between 5' of 'R and the Pvu-11 site by the following method. inserted into a plasmid.

Plasmid HG I-I 3 IR was digested with He1U II and Su11. tac promoter and Bgll
HGfI5 encoding mature HG H up to the I site
A fragment containing the 'DNA sequence was isolated.

Plasmid I-(C; t42.07-1 was digested with 屡II and PSLI.BglII-PstI containing part of flGH and part of P-24.
The fragment was isolated. Ps the same plasmid
Digested with tI and s±aI and then isolated the fragment containing P-15 and part of P-24.

The three isolated fragments were ligated to construct pf(GHP 24-B). This plasmid was fused to the P-24 core protein and P-15 protein, 14Gf(GHP24-B), under the control of the tac promoter. 1) Contains the NA sequence for expressing mature HGH, with the C-terminal 8 amino acids of HGH removed.

pHGHp-24B was digested with HindIII and then ligated to yield the same deletion as obtained for p241)E. However, this generated sequence is a fusion polypeptide of the mature HG f( (Nine C-terminal amino acids are missing □□
□ N / ] is expected to occur. This plasmid is p
HG Hp -24Δ1]D3.

Example 2a This example demonstrates the isolation of a cDNA encoding the gp41 envelope protein, its repair for expression in prokaryotes, the expression of a p41 fusion in E. coli, and the in vitro reproduction of the expressed fusion. Disclose solubilization.

Isolate viral mRNA, prepare cl)NA from the viral mRNA, and inject this cl) into λGTIQ phage as described in Examples IA and B above.
NA was cloned. cl) NA clone H9
c, 53 is the complete p41 envelope region (bP73
36-8992, was identified as containing the cl) NA encoding Figure 2d. H9c, 53 to Kai 22-
1 and ll1ndHI to isolate fragment I corresponding to nucleotides 7417-7722 of the proviral genome. Fragment I is VaI
Amino acid residues denoted by GInAlaArg and A
snTyr-, encoding the C-terminal residue designated rbrSer. Fragment I was synthesized as described in Example IBI above, with a phosphorylated blunt end and a 5' hydroxy EcoRI end, and a υRI adapter (AATTCATGTCTTAσCT
326b, which was generated by ligating an excess of 326b (having the sequence CAAGG) and digesting with HindIIl to cleave the dimer generated by the ligation.
pEco RI-HL cells were isolated on a 6% polyacrylamide gel.

Plasmid pNCV (55a)
The vector fragment was recovered by digestion with dIII.

The 326 bp fragment, the fragment encoding gp41, was ligated to the pNCV fragment and the ligation mixture was used to transform Escherichia coli (A, TC
C31446) was transformed with tetracycline. Tetracycline resistance plasmid (PtrpLE
gp41) was recovered. This plasmid is shown in Figure 8A. This reading frame under the control of the trp promoter (
open reading frame) is E. coli trp
The amino terminus of the E gene product has 190 residues at the amino terminus, which is a sequence connected to its leader with an amino acid sequence encoded by an adapter, and 14 residues encoded by the remaining pBR322 sequence. Residue C
-encodes the gp41 fusion with the end. This fusion contained a total of 306 residues.

g l) 41 fusions are amino and carboxy fusions of prokaryotic peptides with gp4] fragments, but delete either or both of the prokaryotic sequences, or other prokaryotic, eukaryotic or synthetic It will be understood and understood that substitutions may be made in the polypeptide. No immunoreactive material could be identified in the cell culture extracts when direct expression of the DNA encoding the gS1 fragment was attempted, rather than as an amino-terminal fusion. The reason for this is unknown, but may be related to the instability of the unfused product in E. coli. Therefore, g p 4.1 or a fragment thereof should be expressed as an N-terminal fusion in prokaryotes. Such fusions would, of course, include fusions with veliplasmic or extracellular proteins of E. coli, such as, among others, the secretory leader of ST-II thermostable enterotoxin or alkaline phosphatase. In this case g p 4.1 would be secreted as a mature protein lacking prokaryotic sequences. A plasmid encoding a fusion of the signal peptide and the mature gp41 fragment is constructed using appropriate double-sided restriction sites and adapters or linkers if necessary.
Synthesis 1) encoding a known prokaryotic signal sequence in place of the adapter sequence
Easy to assemble by inserting this. Any remaining extraneous molecules, [l)NA, are deleted using M13 phage deletion mutagenesis or other conventional methods.

In particular, sequences near the amino and carboxy termini, two long hydrophobic chains of 28 and 22 amino acids contained within the putative transmembrane region of gp41, are particularly rich in hydrophobic residues. To avoid this, the tip of gp41)NA was truncated to create a 102-residue fragment.

Hydrophobic regions are sometimes known to be detrimental to E. coli growth (56,57). The region spanning residues 29-130 was relatively free of such hydrophobic regions compared to the amino terminus of gp41.

However, it should be noted that other host cells such as mammalian cells are suitable for expressing the intact gp41 protein or gp41 fragment containing 10,000 or both terminal hydrophobic regions.

Large 111% J'4 (ATCC:31446) p
Transformed with t r Pr-Egp'l 1 and A3
Grow in M9 minimal medium until 5o values of 6.6.9.12.17 and 21.

Total cellular proteins were separated by electrophoresis on a 13% 5I)S polyacrylamide gel and stained with Coomassie blue. The results are shown in Figure 8B. In this figure, lanes a-e correspond to the order of increasing density, with lane 1 representing total cellular protein from a control transformed with pBR322 (k stands for 1000 Daltons). The transformant has an apparent molecular weight of 33,000, consistent with the predicted size of 306 amino acids.
It mainly accumulated proteins that are Dalton (L
Egp41 approximately 10% by weight of total intracellular bacterial protein].

This recombinant LEgp41 was extracted from the protein-enriched refractor by gel permeation and ion-exchange chromatography using methods previously disclosed for the υ''I-E-foot-and-mouth disease virus polypeptide fusion (55a). Purified. Specifically, cells were incubated with 10 mM Tris-HCe, 1 mM
Ei) Washed in TA, resuspended in 4 volumes of the same buffer, sonicated, Corvall G
Refractive cell bodies were collected by centrifugation for 30 minutes at 5.00 rpm in an SA otor. The particles were treated with 7M guanidine hydrochloride, 0.1% β-mercaptioethanol (B
M'E) was washed twice and dissolved. The generated extract is
4 hours in Beckman Ti5Q rotor, 19.0
It was clarified by processing at 0 rpm. The high-speed supernatant was treated with Sephacryl S-300 (5ephacryl S-30
0, passed through a Pharmacia column. LEgp41
SI)
Identified by S-polyacrylamide gel electrophoresis,
It was collected and extensively dialyzed against 7.5M urea, 0.1% BMH. This collection was divided into 1) EAE-52 cellulose
Above, 7.5M urea,
0.1% BME, 10mM Tris-I (Cz (pH 3,
5).

The LEgp41 substance flowing out from the column was collected and 0.21
4M urea, 10mM Tris-1 at a concentration of ngSAng.
'-TCP (ptT8.5) dialyzed fl. After purification and silver staining on S-polyacrylamide gel, Egp41 was 95mm and 12mm uniform. LEgp4] was not cricosylated.

This purification procedure uses the LEgp41 fusion to
l l) Eliminates the need to preadsorb patient serum with Escherichia coli protein to assay the amount of S antibody.

Apparently there are little or no pre-existing antibodies against the LEg moiety in the patient's serum, but at least
The antibody is not in the same conformation as in p4.1. This is despite the fact that humans are commonly exposed to bacterial proteins; , which also implies that the risk of inducing a hyperimmune response is low. Prokaryotic protein fusions for use in vaccines were identified by screening pooled sera from normal individuals for the presence of antibodies that cross-react with the candidate fusions. Western plotting (We s = ter
Conventional methods such as n Blotting) or radioimmunoassay were used for screening.

Example 2b Purified LEgp4] (Example 2a) as described (58)
0.05M sodium carbonate (pH 9,6) as
The serum was diluted 1:100 with TBS and applied to a 96-well (well) microvolume (titer) assay plate.
Incubate in the smeared microassay well for 1 hour at room temperature. After washing three times with TBS, the plate was washed with antibody-enzyme conjugate (mouse monoclonal
Anti-human IgG Fc-horseradish peroxidase, Travenol-Genentech Diagnostics (Tr, avenol-Genentech 1)
diagnostics)] for 2 hours at room temperature. Wash the plate again with TBS, 3.
The color was developed with 3', 5, 5'-tetramethylbenzidine, and the chromophore was measured with a 590 nm plate reader (Dynachiku, ]ynaLech). Samples with absorbance values greater than or equal to twice the average value of the negative control serum were evaluated as positive.

Significant amounts of antibodies against purified LEgp4
It was detected in 125 cases (98%) of the serum of 127 patients clinically diagnosed with A11)S. In contrast, commercially available whole-virus ELISA test materials were used to
Sera obtained from 300 random hemoglobins known to be seronegative for V-III had a truncated absorption value exceeding (01)590)0.12).
gp4] was not observed at all.

Two cases of A l l that did not react with LEgp44)
The serum of the S patient was the only AII) S serum that did not react with the commercially available all-virus ISA. One of the sera reacted with whole virus in the Western blot, which can be attributed to a reaction with the P24 core antigen. Another case of LEgp41 negative A
11) S serum did not react with virus in Western blot analysis, but virus could be cultured from this patient. LEgp41 and 125 cases of positive A
II) Reaction with S serum was 0 in only 3 cases. I) 590
The value was <0.4 unit, but 0.21-1.640D
It was in the range of 590 units and was very sensitive. Thus, recombinant LEgp41 exhibits excellent specificity and sensitivity for the detection of antibodies against AIDS retroviruses.

Similarly, by purified LEgp41 ELISA, ATt
Antibodies found in the serum of patients with C (AIDS-related complex 1) and healthy homosexual men were detected.

Of the sera from 69 ARC patients, 60 (86%) were LE.
gp41, and obtained results virtually identical to the commercially available whole virus ELISA. Of the 9 non-reactive ARC samples, 2 sera were positive only for p24, while the remaining 7 sera were completely non-reactive, although virus could be recovered from 4 of the patients. It was revealed by Western blotting using whole viruses that the virus was sexually transmitted.

As expected, a small portion (26/75.34%) of serum obtained from asymptomatic homosexual men
1 ELISA. 2 of this group
6 cases of LF, gp4] The ELISA-positive sera included all of the 24 positive sera detected by the commercially available whole-virus ELISA, and were also positive by the Western blotting method using whole viruses. Importantly, two of the LEgp41-seropositive samples obtained from healthy homosexual men
An example is that it was found to show reproducible non-reactivity in a commercially available all-virus ELISA.

In other serological tests, diseases characterized by false positive reactions [systemic erythematous lupus, rheumatoid arthritis, monomu-cleosi
Goodpasture or Goodpasture
- sera obtained from six patients with urinary syndrome]
There was no evidence of non-specific reactivity in the LEgp41 ELISA+. Furthermore, as a test of specificity, sera that had previously been falsely positive for all commercially available viruses, E1 and 15AI, were tested for LEg.

41 tested by ELISA. Commercially available all-virus ELI
The 58 serogroups that showed irreproducibly positive results in SA were L
Egp4] It was found that there was no reaction in ELISA. Furthermore, 25 sera that showed reproducibly positive results in a commercially available ELISA test but negative results in a more precise Western blot assay also showed LEgp41.
There was no reaction in the ELISAI.

Example 3 Immunoassay of expressed recombinant polypeptide Example 2
E. coli 294 cells containing each of the plasmids indicated were initially cultured in tryptophan-enriched I-broth. This medium represses the trp promoter. After the O,D of the L broth culture is approximately 0.5 at 550 nm, expression of the recombinant polypeptide is induced by transferring the culture to M-9 medium without tryptophan. After about 1 hour, indole acetic acid was added to the medium to give about 1 hour.
A final concentration of 0 μg/Ie is used to induce further expression.

After an additional 5-6 hours at 37°C, cells are collected by centrifugation, lysed (47) and subjected to Western blot analysis.

Immunological reactivity of the polypeptide expressed by the plasmid identified above was determined using Triton-treated IIT LV.
-II inoculated with retrovirus and subsequently live II
The assay was performed using rabbit serum boosted with TLV-III virus.

Expressed in M-9 medium supplemented with indoleacetic acid, I-
TTr, V-ITI rabbit antiserum and 125
I-labeled protein A (Protein A,)
I reacted. I) 241) E and P24+
) E△1 (D3 polypeptide, as well as pl' (GHp
A Western plot of the 24 B and plIGHp 24Δ...)3 fusion polypeptides is shown in FIG. P24
I) Multiple immunologically reactive bands against the E polypeptide appear in lane 3.

The band corresponding to a molecular weight of about 44,000 Daltons is P-
24 and P-15. Surprisingly, the number is around 24,000 and 15゜000.
Two doublets of molecular weight were also observed. The cause of these double line signals is currently unknown. However, the g produced in the naturally occurring polycistronic gag region
The detection of these doublet signals with molecular weights consistent with ag proteins indicates that this E. coli strain is capable of processing such precursor polypeptide sequences and detecting them by complementary antibodies. ■This shows that it is possible to produce I protein. p −24,
A Western plot of DE'D3 polypeptide is shown in lane 4 of FIG. Again, about 20,00
A doublet with a molecular weight of 0 Daltons is observed. P2
41) 44. detected for E. ．． The P-24/p-15 full-length HT IV-III protein of 000 daltons and the lack of a doublet of 15.000 daltons
Consistent with the description of the p24DE Western plot above.

Lanes 5 and 6 of FIG. 6 contain proteins that are immunologically reactive expressed by pHGHp24B and pHGlip24ΔH1)3, respectively. The band in lane 5 with a molecular weight of approximately 65,000 Daltons corresponds to the expected assembled constituent polypeptide. In addition, the lane 6 band is about 4.
It has a molecular weight of 5,000.

Assay with human AII)S serum The polypeptide expressed by plasmid P241)E was assayed for immunological reactivity with the serum of individuals suffering from AII)S. In FIG. 7, strip 3 represents a Western plot obtained from human AII)S serum and P241)E polypeptide treated with 125I-labeled protein A. Approximately 24,0
A wide band with a molecular weight of 0.00 Daltons is seen.

Strip 2 is a control containing normal human serum and p-24DE polypeptide treated with 125N-labeled protein A. Strip 1 contains size markers. This assay shows that the polypeptides of the invention can be used to detect complementary antibodies produced in response to exposure or infection with AII)S-associated retroviruses.

Vaccines Vaccines of the invention are contemplated and contain the predetermined polypeptides and fusion polypeptides described above. The next example is HTI-V'-II
Discloses the use of vaccines containing P-24 polypeptide sequences derived from P-24. However, AII)
Any polypeptide sequence of an S-associated retrovirus, particularly that encoded by the env region of the retroviral genome, may be used.

Example 4 Immunization of Mice The polypeptides encoded by P24DE and P-24DEΔHD3 are used to immunize BALB/C mice. Emulsion consisting of 50% aqueous antigen and 50% complete Freund's adjuvant 2
5-25μq of P-241)E contained in 00μl
or P-241)EΔ[-11)3 polypeptide. Each mouse is immunized at multiple epidermal and subcutaneous sites (i.e. 25 μl on the sole of each hind paw, 50 μl on the tail and 100 μl distributed over 3-5 epidermal sites along the spine) as follows: Control. Groups are similarly injected with an emulsion containing no antigen.

- Four weeks after the secondary immunization, mice are boosted with 5-25 μg of polypeptide as described above, except that the emulsion is prepared using incomplete Freund's adjuvant. Boosting immunization is performed by giving each mouse 200 μl of emulsion with or without antigen, distributed as follows: 50 μl in the tail and 50 μr distributed in the epidermis along the spine. Three weeks after the booster immunization, approximately 500 μl of blood is collected from each mouse by tail bleed. Serum obtained from this blood is used for in vitro neutralization studies.

In vitro neutralization studies The ability of sera obtained from mice immunized with the P-24DE or P-241)EΔH1)3 polypeptide and from control mice to neutralize I-TTLV-III was determined.
Test in vitro. Serially diluted mouse serum (
2-fold dilution, 1:8 to 1:16384) 25μ
1 ulbecco's improved eag/l/ with HTLV-III concentrate 175 ties.
(Incubate for 1 hour at 37°C in Eagl medium inlet. After incubation, each dilution is
Approximately 40,000 HUT-73T- cells (48) were placed in each well of a tissue culture plate with 6 wells.
Co-application-J-Rule.

After 3-4 days of incubation, viral growth is determined by immunofluorescence assay for HTLV-nI (Immunofluorescence assay) or reverse transcriptase assay. I-ILIT-
The lack of LIT IV-III infection of 78T-cells indicates that the polypeptide was isolated from I-TT L V-II in mice.
This shows that the production of neutralizing antibodies against I was induced.

Neutralizing antibody assay in primates Using a polypeptide that generates neutralizing antibodies in mice, we conducted a test using polypeptides that generate neutralizing antibodies in mice. Immunize chimpanzees. Each chimpanzee in the experimental group is immunized with 50%
immunization with 50-100 μg of polypeptide contained in 200 μl of an emulsion consisting of aqueous antigen and 50% complete Freund's adjuvant. Each chimpanzee is immunized with multiple iron site inoculations as described below. The control group is injected with an emulsion containing no antigen. After 4 weeks, each group of chimpanzees was treated with an emulsion similar to that described above, except that it was prepared in incomplete Freund's adjuvant, or with a 200% antigen emulsion.
Promote immunization with μl.

After 5 weeks of boosted immunization, chimpanzees in the experimental and control groups were challenged with various I(TLV-III concentrates). It is a candidate vaccine that can induce the production of neutralizing antibodies in humans that resist infection by retroviruses.

Complete Freund's adjuvant, which is not tolerated for use in humans, was identified to inhibit lymphadenopathy in chimpanzees challenged with IITLV=lII.
The above polypeptide was added to an auxiliary solution suitable for human use (
Formulate with adjuvant.

Such a formulation may contain an alum precipitated polypeptide complex (22) used to immunize the experimental group of chimpanzees. The control group will be vaccinated with the auxiliary solution alone. The formulation inhibiting lymphadenopathy in the experimental group contains the polypeptide in a mixture with pharmaceutically acceptable excipients which may be used as a human vaccine.

Example 5 pHGHp-24-B and pHGHp24ΔHD3
Example 4
I-ITLV-III
Determine which constituent polypeptides confer resistance to AII)S infection in chimpanzees, as evidenced by the lack of development of lymphadenopathy in immunized chimpanzees challenged with retroviruses. However, the use of IIGH polypeptide sequences within such constituent polypeptides has implications for AIDS, as such constituent polypeptides may induce autoimmune responses against f-I G II in human subjects. It is not preferred in human vaccines against infection by retroviruses. Therefore, a vaccine of constituent polypeptides for conferring resistance to infection by AIDS-associated retroviruses in humans would be virtually impossible to elicit an autoimmune response against naturally occurring polypeptides in humans. AII) S-associated retrovirus, or a fragment thereof, expressed as a fusion polypeptide with a polypeptide sequence of AII).

A I r) S-associated RNA dependence 1) A polypeptide sequence of NA polymerase (AII) S reverse transcriptase 9 that suppresses the activity of such a transcriptase and
It is contemplated that the assay may be used to identify compounds that can be used as drugs to inhibit infection by related retroviruses, or dissemination of the retrovirus in infected individuals. Suramin (53), known as an inhibitor of AII)S reverse transcriptase, will be clarified in the following example. However, since suramin exhibits severe clinical side effects (53) when administered to humans, other transcriptase-inhibiting compounds will be tested. Examples of such selective inhibitors have been demonstrated by several laboratories (54).

Example 6 A, AII) Cloning and expression of S reverse transcriptase ■
fa[ of the viral genome encoding the AII) S-associated retroviral reverse transcriptase of TLV-III.
ends at nucleotide 4674. Its N-terminus is
It is located within a region extending from nucleotides 1639 to approximately 1800. For the purposes described below, the gag region (1639-1
769) It is preferred to choose an internally located amino terminus. The region adjacent to nucleotide 1800 contains a number of suitable fjmet codons useful as initiation codons for constructs expressing reverse transcriptase. However, it is not important to choose the native met codon. Instead,
The exogenous ATG codon carried by the vector is
Ligate to a partial exonuclease digest or MI3 deletion mutant of the 5' region of the reverse transcriptase gene. Constructs assembled correctly in frame are readily identified by their ability to reverse transcribe RNA of transformant cell extracts.

Alternatively, the N-terminus of an in vivo normal reverse transcriptase, as processed in a particular host, can be purified from infected cells and the amino terminus sequenced according to methods known per se. c encodes the amino-terminal amino acid sequence
Determined by identifying the DNA nucleotide sequence. Preferably, the cDNA of the figure is nucleotide 1642
Cut with φh,! -II, and emesis I (5367)
Digest it with. A 3725 bp fragment is recovered. The N-terminal cleavage site other than SHII is 1738 (hereinafter referred to as 1e
I) and 1754 (Alul). However, these enzymes also cut at locations internal to the reverse transcriptase gene. Therefore, in order to recover the full-length gene, D
Partial digestion with dI or AluI must be performed. The other cleavage site other than Sal■ located distal to the reverse transcriptase gene is Stu I (located at 4987).

The DNA encoding the reverse transcriptase gene with terminal ligation sites is isolated and then ligated into a replicable vector. Vectors are selected depending on the intended host, but
This depends on whether 1) the NA is simply to be replicated or one wishes to use the vector for expression of the enzyme; Typically, the vector contains a bacterial origin of replication and an antibiotic selection gene, such as tetracycline resistance. These elements are p
It can be obtained from a number of commonly known plasmids such as BR322.

Since it is preferable to express the reverse transcriptase in higher eukaryotes, the vector can be used for transcription of the gene in higher eukaryotes for stable replication of the gene, for the identification of transformants. It also contains the elements necessary to promote transcription and to properly terminate transcribed mRNA. Representative examples of these elements include the SV40 origin of replication and the T antigen source [Cold Spring I (a
The gene encoding mouse thymidine kinase, SV, is supplied by chromosomal integrants found in cell lines such as CO5-1, available from RBOR Laboratories).
There are 40 early or late promoters and polyadenylation sites for the hepatitis B surface antigen. Such vectors are known (e.g. cited in EPA 73°656).
．． 92.182 and 93,619).

Known vectors may not contain convenient restriction sites for direct insertion of the reverse transcriptase 1) NA obtained as described above. This will not be a hindrance to the person skilled in the art since methods of introducing new restriction sites or converting sticky restricted ends into blunt ends are known per se.

For example, the pH5 indicated in EPA 73,656
The hepatitis surface antigen gene in 94 was dissected out with EcoRI and poor tII digestion, ligated into the BglII and SalI sites of the coRI and BamHI linkers, respectively, of the 1)NA fragment, and this modified cl)NA (594). This assembly is then completed according to EPA 73,656.

The vector carrying the reverse transcriptase gene is CO5-1
Transfect into a permissive host, such as the monkey kidney cell line (cell line 1) or the C11O (Chinese hamster ovary) cell line. Other stable eukaryotic cell lines may be used as hosts, as well as suitable yeast and prokaryotes [for yeast, micron replication origins and promoters such as metallothionein (7) promoter 1-5] for bacteria, pBR
322 origin of replication, and A11) t as described elsewhere herein in connection with the expression in prokaryotes of the gag and envelope proteins of S-associated retroviruses.
promoters such as rp] may also be used.

In this regard, other AIDS-associated retroviral proteins such as gag and envelope polypeptides
It can also be expressed in eukaryotic cells, which have a closer phylogenetic relationship than prokaryotes to the usual retroviral hosts, whether yeast or mammals.

Preferably, the reverse transcriptase is synthesized in recombinant culture under the control of an inducible promoter so that any toxic effects of the polymerase on the cells are minimized until large quantities of rn RNA have accumulated.

Alternatively, a signal sequence that is recognized and processed by the intended host (e.g., in the case of higher true, plant life,
The gene encoding the enzyme is ligated at its 5' end to a moon that contains a known secretory signal sequence for a secreted hormone such as interferon, insulin, or a viral surface antigen.

B. Assay of reverse transcriptase inhibitory effect AII) The cell culture expressing the reverse transcriptase of S is lysed and divided into equal amounts. Various amounts of the test compound are added to each aliquot of the lysate. The poIJ A oligonucleotide is added to each aliquot along with oligo dT primer and 32p labeled TTI'. After incubation at 37°C, acid precipitation counts are determined.

Next, inhibit the reverse transcriptase of AII)S.
) Compounds that do not inhibit NA polymerase are screened for further in vitro toxicity and efficacy studies.

Alternatively, the reverse transcriptase of AI DS can be isolated by methods known per se, including immunoaffinity adsorption, Sephadex gel filtration, ion exchange chromatography and isoelectric focusing on untreated polyacrylamide gels. from the expression host. The recovered reverse transcriptase is then used to assay compounds as described above.

Example 7 Cloning and Expression of the E' Polypeptide A I I) The E' polypeptide of the S-related retrovirus is designated as 1E' in Figure 2d.
a polypeptide of a residue, a naturally occurring allele thereof,
or AIDS-associated retroviruses are defined as amino acid sequence variants thereof that have immunological cross-reactivity with antisera capable of binding to the E' polypeptide produced in infected cells. In addition to the C-terminal deletion derivatives described below, do not substantially alter the immunological reactivity of the peptide with serum from patients infected with 1-ITL V-Ill or other AIDS-associated retroviruses; Other deletions, insertions or fffiJJ derivatives are also referred to as "E'polypeptides" or "E'proteins" as used herein.
Included within the scope of the term. Yet another example of a deleted fecal variant is the E' protein, in which the first 19 residues up to Arg19 are deleted. - The trLE fusion described in
It is an insertion mutant. Other variants will be apparent to those skilled in the art. These can be easily produced by recombinant synthesis;
Certain deletions consisting of or can be produced by proteolytic hydrolysis.

According to the present invention, the E' polypeptide can be produced by recombinant methods so as to be free of proteins derived from AIDS-associated retrovirus-infected cells that are not encoded by AIDS-associated retroviruses. Such polypeptides are apparently not present in virions of most AIDS-associated retroviruses.

cDNA clone H9, c53 contained the E' sequence. This E' sequence is shown in Figure 2d as the sequence starting at nucleotide 8375. The first stop codon in the open reading frame downstream of ATG at position 8375 is the OP stop codon at nucleotide 8993. The Cys residue just before this stop codon is likely the C-terminus of the E' polypeptide. However, other C-termini upstream from this Cys residue are also within the scope of the invention.

The E' polypeptide comprises a sequence C-terminal to any of the last approximately 50 residues shown in Figure 2d, preferably downstream of and adjacent to a lysine or arginine residue. A sequence with the group at the C-terminus is preferable.

An expression plasmid for synthesizing a fusion between a bacterial polypeptide and a bacterial polypeptide is shown below.
I will write down this. 89. Haell a part of c53
and Xho), and a 62 bp fragment encoding E' residues 14 to 34 was recovered (fragment 1). A separate sample was digested with Xhol and nd111 and a 71.9 bp fragment was recovered encoding amino acids 35-206 and containing the stop codon followed by untranslated 3' sequences (Fragment 2).

Sono 5' end 1cEcoRJ viscous end, l-1a
e III' i A sequence as follows encoding the first 13 residues of the E' polypeptide, ending with the 3' blunt end for ligation to the blunt end (blunt end) of fragment 1 generated from: A synthetic oligonucleotide (fragment 3) was prepared with (35).

p N CV (55a), both p N C
EcoR■ and Hindl, the only sites in V
II and the vector fragment was recovered. This vector fragment was simultaneously ligated with fragments 1, 2 and 3, and the ligation mixture was introduced into Escherichia coli (E, coli) 294, and antibiotic resistant colonies were identified. From the preparation of plasmid DNA, ptrpLE-E' was obtained from a resistant colony.

The structure of this plasmid is shown in Figure 93.

ptrpLE-E' is a complete E' at the C-terminus.
Bacteria (E. coli) fused with the sequence
) contains a continuous (open reading frame) encoding a protein derived from the protein, white protein (L E ).

This plasmid was transformed into an E' protein fusion (L
, E-E'). Escherichia coli (E, col)
1) The 294 strain was transformed with ptrpLE-E' and grown overnight in M9 broth containing tetracycline (32) containing 5 mcg/me of tetracycline. Dilute 50i,
The cells were grown at 37° C. until the absorption at 550 nm i was 0.5. Total (whole) cellular protein was prepared from 25 ml of induced cell culture. cells, 0.01M
Tori Sue - HCj I (p+(7,5), 0.00
1 M E DTA, 0.03 M mercaptoethanol and 0.8% SI) 1/250 of S. 11
) The carp was clouded, boiled for 2 minutes, and precipitated with 3 portions of cold acetone. S'1) The precipitated proteins were redissolved by boiling in S-polyacrylamide Kelp buffer (40).

Initial attempts to directly express the unmodified 1 comparison sequence in E. coli failed due to the apparent instability of the protein. However, the method described above resulted in satisfactory expression of the E' polypeptide in E. coli. LE is a 190-residue protein derived from the translated sequence of LrplJ-gu and the trpE gene product (55b), which forms stable insoluble aggregates in vivo and is expressed in E. coli (E.

It has been successfully used to stabilize the synthesis of other foreign proteins in E.coli)i (55c, 55a).

Expression of the I-E-E' fusion protein from p trpLE-E' was performed using the Escherichia coli (E. coli) trp promoter and the LrplJ-guribosome binding site (first
Figure 3). A culture of Escherichia coli (E. coli) transformed with ptrpLlOtori-E' was
To derepress the promoter,
Grown under butophane-starved conditions, the protein was reduced to 10%.
Electrophoresis on 5O8-polyacrylamide gels and staining with Cuman-Blue [therefore analyzed]. Chapter 8b
As shown in the figure, cells containing the plasmid ptrpLE-E' accumulate a significant amount of Mr41,000 protein (column b), which is associated with E. coli transformed with PBR322i. ) is not present in the culture (column C). Mr41,000 protein is expected to be sold 1
The ゜E-E' fusion protein has two important features. First, this protein is serologically reactive with antiserum to L E and, second, it is a size match that would be expected for a protein containing additional amino acids of Sala1co206. 22,000 larger than I-E ($81 diagram, column 3).

A I I) After being attacked by S retrovirus, LF, E
To examine whether '' represents an antigenic site recognized by human serum, total cellular proteins extracted from Escherichia coli (1': coli) transformed with pLrpLE-E' were analyzed by Western blot analysis. Used as antigen in the assay. Individual sera were collected from Escherichia coli (Ii).
It should be noted that preadsorption was done with a =T-soluble extract (blocking extract) of the E. coli ) proteins, but this was later found to be unnecessary.

In Western blot assays, total cellular protein was prepared from amplified cultures of E. coli transformed with ptrpLE-E' (Figure 8a) or ptrpLE-gp41 (described above). ,
Electrophoresis was performed on a 5DS-10% polyacrylamide gel as described above. Proteins were electrophoretically transferred to sea urchin nitrocellulose paper (sort) as described (55e). 5
% normal goat dish serum and protein-blocked extract from E. coli transformed with p13R322 5 m
TBS buffer (0,025 N4 tri:z, -F (Cl (pH 7,2), 0.15 M N
1:2 in aC1 and 0.05% Tween 20)
Individual plot strips were incubated overnight at room temperature with the indicated serum at 0.00 dilution. The blocking extract removes bacteria from 0. OIM') Lis-1 (Cβ (pH 7,5),
pNCV or p
Escherichia coli (E, col) transformed with trpLE-E'
i) Total cellular protein prepared from L E or L
It was used for preliminary adsorption of E-E' protein and serum. T.B.
After thorough washing in S buffer, the paper strips were treated with biotinylated goat anti-human or anti-rabbit TgG (Vector Labs), horseradish peroxidase conjugated with avidin (
Miles-Yeda)
and the peroxidase substrate (4-chloro-1-7tollQ in TBS,
5 nll/me and hydrogen peroxide 0.16%). After each incubation, remove the paper strips in TBS.
I washed it carefully.

37% AII) S patients (17/46), 81
Test sera from 3% of ARC patients (21/26) and 39% of healthy homosexual men (29/75) were LE-
showed a clear reaction with E/Immunoplot, whereas no reaction was found with any of the 37 sera from randomly selected blood donors (Table 1 below). ρtrpL E
-F' derivative plasmid ptrpLE17-E
Identical results were obtained for a representative collection of these sera in Western blot experiments using total cellular protein from E. coli transformed with E. coli as antigen. These cells contain LE proteins 1-17.
Expression L, A of I-E17-E', a fusion protein that differs from LE-E/ in that only the residue
II) The serological reactivity detected between S-related sera and LE-E'i binding proteins indicates specificity for the epitope associated with the E' sequence.

F-': LE-E' reactivity in western blot assay, gp41: reactivity in LE-gp41 western blot assay (see example above), WJ3: whole virus western blot assay Reactivity in HHM: healthy homosexual men, control: random donor sample.

``This individual was positive in the I-T' IV-111 culture assay.

(])) All 37 control sera were commercially available all-virus ELIs.
He was seronegative in SA (Abbott).

Different A I I) This LE-E' in S risk group
Two important points are clear from the comparison of gp41 and gp41 seroreactivity. First, L E -E' seroreactivity is detected much less frequently than gp41 seroreactivity in AIDS patients (37% vs. 96%).
) are about the same frequency for sera from ARC patients (81% vs. 85%) and healthy homosexual men (39% vs. 32%). Second, a significant number of healthy homosexual men were seropositive for LE-E' (r117) but not for gp41 (11/75). Antibodies against E'J were found in a statistically significant number (11/75) of healthy homosexual men, namely AII)S and AItC, as well as in a smaller number of AIDS and AFLC patients (3/72). The fact that antibodies against env antigens are detected more rapidly than antibodies against env antigens in a group at high risk of developing
This is completely unexpected and surprising since this protein has not been detected as a virion component, and it was unexpected to find this protein in the serum since E' does not contain an obvious secretory leader. Ta. Seroconversion against env antigens was performed using all HTLV-J viruses (55e
) or recombinant LE-gp41 antigen (described above). Each assay has >98% reliability in detecting serum from clinically diagnosed AIDS patients.

Because many of the healthy homosexual men studied here were seronegative for E' as well as viral antigens, and therefore would not have been exposed to the virus, the E'mo gp41- individuals in this sample The actual ratio is 31% (
The rate may be as high as 11/35). These results are related to the previous finding that AIITL V- and can be isolated from lymphocytes of asymptomatic seronegative humans (55m), and are useful for early clinical diagnosis and diagnosis of AIIS retrovirus exposure. It may have considerable practical importance for screening.

In contrast to the situation found in asymptomatic individuals exposed to the virus and in ARC patients, antibodies against 1likelihood were detected much less frequently than antibodies against gp4 env in patients with AI I)S. (37% vs. 96
%). A similar pattern of low frequency reactivity of S-thoughts has previously been highlighted for the nuclear (core) protein p24gag when compared to the major tegument protein gp120env in radioimmunoprecipitation analysis (55
n). Although it is not certain whether this present case represents a unique susceptibility that develops into A I f) S, this suggests that E' and P24
This is more likely to reflect a selective reduction in antibody titers to gag or lower antibody titers.

To obtain direct evidence of E' expression in H-rLV-III infected cells, high titer antisera specific for E' were isolated by preparative Sf) S-polyacrylamide gel electrophoresis. was prepared by immunizing rabbits with LE-E'. The gel portion containing L E -E' (approximately 100 mCg) was scraped off, homogenized in Freund's incomplete adjuvant, and administered intradermally at two week intervals. E'-
This rabbit serum was screened for its ability to immunoprecipitate relevant proteins. For this purpose f1, commercially available (7
) H9/ I I T I-V-] [B (Rulai 7
It was used. This is because the present inventors believe that this cell is E′
1, which has the potential to encode translation products.
7-1.9 kb mRNA (~1% polyA+RN
A) was previously observed to produce high levels of A) (55e). After four antigen injections, serum obtained from two rabbits was 19/HTLV-Jl[
5-6 distinct Mr23,500-28 from B cells,
000 proteins could be immunoprecipitated. Preimmune serum obtained from the same animal did not immunoprecipitate any of these proteins. Furthermore, this immune serum did not detect any of these proteins in uninfected H9 cells. r-19/t(T L
The protein detected in V-1111B cells was M
Major proteins of r 28,000.25,500 and 25,000 and M'r 26,500 and 23
, 500, and contained a small amount of protein. In some experiments, the Mr23,500 protein has two distinct
It was possible to divide it into different types.

LE-E' positive sera from three ATI) S patients and one ARC patient were i(TLV-II
We were able to immunoprecipitate ~Mr28,000 and 25,000(7) proteins from I-infected cells that were not precipitated by serum from control blood donors. However, immunoprecipitation of these proteins is only partially presented by L E -E' proteins, and L E -E' to A I
1) Suggests that S and ARC sera recognize the E' antigenic determinant, which is not presented by the recombinant protein.

This rabbit antiserum contained antibodies capable of binding to the E' polypeptide of the AII) S-associated retrovirus, but did not contain antibodies capable of binding to any other polypeptide encoded by the AIDS-associated retrovirus. Furthermore, it did not contain any bound E' polypeptide.

Usually immobilized to facilitate separation. For example, the antiserum or antibody is bound or adsorbed to the microtiter plate holes of a polyolefin, such as polystyrene, or to a matrix that is preabsorbed with anti-rabbit TgG (eg, goat).

This E' protein was then expressed in recombinant mammalian cell culture. Our starting plasmid was obtained from pFDll (E'P117.06OA) by a complex procedure using plasmids readily available to us. This method is not preferred for use by those skilled in the art. Alternatively, the starting plasmid pFDll is preferably obtained by the following method. pFI) 1,14, digested with Hind Jll at its unique Hindll [site and recovered the vector.

11: The adapter with the following was ligated to the vector fragment and introduced into Escherichia coli (E. coli) 294. pl'1)lid is obtained from ampinlin-resistant colonies. By ligation of an adapter to pFI)1, this mouse I) B was immediately upstream of the HF R gene.
am14I and C1al sites are introduced.

pFDlld is digested with Bamf-II and C1al and the vector fragment is isolated.

ilgG, 53 with Bam II and TaqI? f
13amH at nucleotide 8053
This human bP fragment (fragment 4), containing the enV gene portion of the I site, the E' coding region and the untranslated 3' region up to nucleotide 232, was released #L. The untranslated 3' region contained 3' long terminal repeats (LTk). The pFDlld vector fragment (described above) was ligated into fragment 4 and E. coli (F., coli) 294 was transfected. pSVE, E'DI-IFR was obtained from an ampicillin resistant colony. In this way, E′ encryption■
) NA is placed under the transcriptional control of the early promoter of 5V4Q, while LTR drives transcription of the dhfr gene (
(promoting) sequence, as well as sequences for cleavage and boriatenylation of the E' gene transcript.

CI (Odh fr-K I DUX-B 11 cells (
55f) containing 10% tallow serum.
The cells were grown in M medium. Transfection with pSVE, E'DHFR was performed (55g) by the phosphate precipitation method as described (55h). After 6 hours of vector exposure, cells were treated with 20% in PBS.
Shocked (55i) with glycerol (v/■) and grown for 1-1/2 days in non-selective medium. Cells were then passed through selective medium (F12 medium lacking glycino, hypoxanthine, and thymidine and supplemented with 10% dialyzed fetal calf serum) and fed with medium every 2-3 days. A resistant colony of approximately 300 individuals was formed in a cluster after two weeks. Immunoprecipitation analysis of one of the initial dhfr(- colonies revealed barely detectable levels of E
' was shown. This population was selected for growth in methotrexate (53j, 55k). 2×105
Cells were plated in 1-00 mT)I dishes containing selective medium containing 10'M methotoxate, and the medium was changed every 2 days. After 3 weeks, this medium (C1-107F, '1
00) A group of resistant colonies of about 50 individuals appeared, and the E' protein was recovered as follows.

Cells were harvested by centrifugation and phosphate-buffered saline (P
B S ) and RIPA buffer [0.05 M Tris-1-IC1 (pH
7,5), 0.15M NaCg, 1% Triton X-1
,00,1,% Deoxocholic acid and H'o,i%
Sodium sulfate] was dissolved (lysed) in 2me. The extract was added to 10 mc of preimmune rabbit serum for 2 hours at 4°C.
Preincubate for 1 hour, clarify twice with 50 mCl of Pansorbin (Calbiochem), and remove rabbit anti-LE-E.
'The serum was incubated with 2mc at 4°C overnight. The immunoprecipitates were co-incubated in Pansorbin Omc for 30 minutes, collected by centrifugation, and the pellet was washed twice with RIPA buffer and once with water. The immunoprecipitate was solubilized and E' was separated from the rabbit antibody by ultrafiltration, electrophoresis or other conventional techniques.

On a commercial scale, the antibody or antiserum is previously insolubilized and E' is eluted from the immobilized immunoadsorbent using a pH 3-5 buffer.

IX 10-7M methotoxate (CHO/E'.

A group of colonies that appeared in 100) produced significant amounts of two E'-encoded proteins that were specifically immunoprecipitated by rabbit serum directed against L E -E' but not by preimmune rabbit serum. None of the proteins could be detected in the immune serum from the CHO parent cells. Furthermore, Cf
(O/E', immunoprecipitation of both E'-related proteins detected in 100 cells was easily competed by L E -E' protein but not by T-E protein;
This fact proves that the reaction is specific for the E' sequence.

By direct comparison, two E'-related polypeptides produced in CHO/E', 100 cells were found to be H9/HTL V
-Mr28,000 and 2 observed in 11 eyes 3 cells
It can be seen that it has the same electrophoretic mobility as the protein No. 6.500. Furthermore, CHO/E'.

100 and r(g/HTLV-IIIB cells) The relative amounts of these two proteins are virtually equivalent. However, 28,000 and 26,500 H9/HT
It is not certain whether the i-V-]1[B cell protein is chemically identical to the E'-related recombinant polypeptide produced here. Pulse labeling studies showed that neither protein in CHO cell culture was a direct precursor of the other. Approximately 80-90% of the Mr28,000 and '26.5001L' proteins are present in the transfected C
It was contained within the cytosolic fraction of HO cells. CHO/
Immunoprecipitation of cell supernatants from E', 100 cultures showed no secreted E' protein.

To remove bacteria, the polypeptide solution was diluted to 0.
The recombinant E' polypeptide is sterilized by passing through a 22 micron filter. The P solution is prepared into a vaccine by optionally further purifying the polypeptide, eg, by gel filtration, and formulating the polypeptide in a pharmaceutically acceptable carrier such as isotonic saline, D5W, and the like.

The amount of E' protein used is sufficient to produce detectable amounts of anti-E' in the subject upon subcutaneous injection followed by an intravenous booster injection. Since this will necessarily result in immunological competition in the test subject, the dose frequency and route of administration of the antigen will be determined by the attending physician through monitoring of anti-E/ levels in the serum. Must be.

E'-containing vaccines optionally contain other previously determined A1. I) May contain polypeptides of S-associated retroviruses.

This will help ensure complete reinforcement of the immune response against potential AI DS-associated retroviral infections.

The role of the E' gene protein in viral reproduction and pathogenesis remains an important unanswered question raised by these studies.

It is easy to detect individuals seroreactive for L E -E' by immunoplot, but not for whole viral antigens, suggesting that E' is not a virion component. However, it is also possible that E' was lost during virus purification. immunoprecipitated by AIDS-related serum, f-
(T L V-11 [- Mr 25 in infected 09 cells
,000-28,000 (55o155n, 55P), and this may correspond to the E' protein, but the same polypeptide is HTL
It was not detected in V-III virion preparations (5
5o, 55P). In CHO/E', 100 cells, most of the intracellular E' is found in the cytoplasmic fraction rather than the nuclear fraction, indicating that E' is involved in nuclear events such as transcription initiation. This suggests that they are not related to each other. However, these studies must be considered preliminary, as they may not accurately reflect the location of E' in infected cells.

Transcriptional mapping studies suggest that E' may be an abundantly expressed protein in infected cells. n g 71-IT t, v -III B
Approximately 20% of the viral RNA synthesized in cells
is a 289 bp sequence containing the sequence from 5' to gag
leader, a central exon located upstream of env,
and 1 from the 3' end of the genome containing the E' gene.
, 3 kb of spliced subgenomic RNA (55e). One of two additional short untranslated IJ-ders starting from the pol or P' domain may also be present.

Two splice song acceptor sites (nucleotides 5.3
Depending on whether (positions 59 or 5,558) is used, either a 268 bp or 69 bp central exon is generated (55e, 55Q). 268hp
The exon contains two AUG-IJ prets that are absent in the 69 hp exon, one of which appears to serve as the start codon for the tat open reading frame (55q).

1, 7-1 containing the central exon of 69 hp
．． 9 k b mRNA1fi oi"'C, the predicted AUG start codon of E' is preceded by one AUG to IJ pret, but the upstream AUG)
The liplet is in contact with an unfavorable nucleotide (55
r), and that the E'AUG initiator is more sufficient than the second-class in-frame termination codon or following this (55S). Therefore, the present inventors believe that the E'AUG initiation codon can be effectively utilized for translation initiation. I was hopeful that I could do it. This -4 second-rate AUG-IJ plate containing plasmid)' (psVE, E
The expectations of the present inventors were confirmed by the efficient expression of E' protein in Cf(0 cells) using 'DHFK). Similarly, which of the two central exons (55e) to use is determined by iat
Determine whether the open reading frame precedes the env gene. If the mechanism for producing mRNA encoding either the E' and env proteins or the tat open reading frame is differential splicing, it represents a critical regulatory event in the reproduction of AIDS retroviruses. Would.

The E' polypeptide or antibodies thereto are used in conventional assays as outlined above.

E' in the test subject's body fluids (serum, saliva, plasma, urine, etc.)
For assays of polypeptides, "competitive" type test kits preferably label E' polypeptides (enzymes,
(radioactive isotopes, fluorescent groups, etc.), antibodies capable of binding the E' polypeptide, and, optionally, other conventional reagents such as incubation and wash buffers. This test kit is suitable for methods known per se for use with selective labels, such as methods using ELISA, radioimmunoassay or fluorescence polarization.
use. Similarly, conventional one-sand German assays are also useful for determining the presence of E' polypeptides or antibodies thereof in a subject sample. Assays for E' protein are uncommon for diagnosis of AIDS. Because this assay does not exist for purified AIDS virions,
This is the first known method aimed at investigating the proteins encoded by the viral genome and clearly expressed by host cells during the course of viral infection and/or replication in vivo. . Detection of such proteins is believed to constitute the first indicator of viral infection, prior to the accumulation of virion antigens such as env and the production of host antibodies against the i(T L V ·-][-encoding proteins. , is the preferred diagnostic approach.

It is also within the scope of the invention to assay body fluids for DNA or RNA encoding the E' protein using standard hybridization assays, such as Southern or Northern assays, respectively.

The DNA or RNA used in such assays need only hybridize to the sequence encoding E' in FIG. 2 and need not encode the E' protein.

Such nucleic acids are easily synthesized as oligonucleotides and then converted into E' polypeptide-encrypted DNA as shown in FIG.
can be screened for the ability to hybridize with. This diagnostic nucleic acid is radioactive phosphorus (1")
Label using a conventional method using a detectable tail such as.

Example 8 Recombinant synthesis of secreted HTLV-1 retroviral envelope proteins in mammalian cells.
major reading frame
It is a figure showing each position. The env reading frame encodes the viral envelope used in this example.

The envelope fragment encoding amino acid residues 61-531 was converted into a secreted envelope fragment.
used to express proteins. This fragment was ligated into a vector containing the necessary components for secretion in mammalian cells as well as for proper expression of the integrated gene. [5v
The 40 oriJ contains the 5V4Q viral early promoter (rEJ), which is used to drive transcription of either the AIDS retroviral envelope or the dihydrofolate reductase (dhfr) gene.

Termination of transcription and message polyadenylation are caused by I
Surface antigen gene of type 3 hepatitis virus (I-I EV
This is achieved using a signal derived from the 3'-untranslated region of the sAg polyA vector). Growth in E. coli is achieved through the intervention of the pBR322 origin of replication and the plasmid's ampinrin resistance gene (rAPRJ). Finally, we transfected Chinese ham sclernoovarian (CHO) cells lacking the dhfr gene to secrete murine dh as a selectable marker.
The fr gene is used.

The method devised for constructing this vector from a generally available starting plasmid is an improvement on the method actually used, as described below.

pE3481-IBV E400D22 (Nmonse 7
(Simonsen) et al., 1983 [Procedure of the National Academy of Sci.
ci,) USAj B□: 2495) was digested with Hpa□, and a linear plasmid was recovered. These Hpa ■ sticky ends were made blunt with Klenow fragment of DNA polymerase ■, and vector fragment ■ was recovered. Code sequence: TCTAGAGGATCCCCAACTAAG
TAAGATCTAG, a blunt-ended double-stranded termination linker is ligated to the vector fragment ■,
After transfecting E. coli 294 with the resulting ligation mixture, the vector is recovered from the AmPr colony. The underlined sequences represent stop codons in each of the three possible reading frames. stop linker
By intervening, a synthetic 9-residue polypeptide is introduced into the C-terminus of the env protein.

This vector attached with a stop linker was digested with Xbal:, blunt-ended with Klenow, and then digested with EcoRl: to recover vector fragment 2 of 5706 bp. This digestion removes the coding sequence of the hepatitis B surface antibody.

HTL V-11 Lutrovirus env cl)N
Hh clone H9c, 53 containing A
Digested with aI, blunt-ended with phre/-, and then digested with EcoRI.
After digestion, nucleotide 5324 [Ec
oRI site] to nucleotide 7394 (e
Fragment 3 spanning nV amino acid 531 (1-1 h a I site) is recovered. Fragment 3 and vector fragment 2 were ligated, E. coli was transfected with this, and vector 4 was recovered from ampicillin-resistant colonies.

Digest vector 4 with Nde■, and the Nde■ site is en
(corresponding to amino acid 61 of the v protein), blunt-ended with Klenow and digested with PstI to recover vector fragment 5. This process converts a single nucleotide located within the AmPr gene into the env protein.
Sequences present up to residue 61 in the 3' direction were removed, resulting in retroviral sogenal and env
The 61 N-terminal residues of the protein will be removed.

pgDtruncDHF'R (EP139,417A)
was digested with PstI and Pvu [3] of the Ampr gene.
' portion (corresponding to the sequence removed from vector 4 in the previous step), the SV40 replication origin, the 25-residue herpes gD protein signal, as well as the first 25 N-terminal residues of this mature gD protein. The 7 fragment 6 of 1514+)p containing the group is recovered. Fragment 5 and 7 fragment 6 were ligated, the resulting ligation mixture was used to transform E. coli 294, and pAIDsenvLrDHFR was extracted from the AmPr colony.
was recovered.

Those skilled in the art will recognize that the vectors described above for expression of p41-viral protein fusions can be modified as appropriate to express polypeptides of viruses other than herpes simplex virus. For example, fusion with hepatitis surface antigen inserts DNA encoding a truncated or intact mature p41 protein in place of the stop codon of the surface antigen, or, alternatively, the starting plasmid This can be easily achieved by using, for example, pF and 348HBV. Selection of appropriate restriction sites and synthetic adapters is within the skill of the art.

Signal sequences other than herpes gDgnal can be used as well. Permissible signals include signals of secreted eukaryotic (preferably non-primate) polypeptides or viruses of mammalian host range, known per se.

The day before transfection and selection, cells from a 10 cti dish full of CHODHFR cells were cultured with F12/DMEMC Delbecco.
Co's modified Eagle's medium], 10% FBS (fox fetal serum), 7GHT (glutamine, hyboxanthene,
thymidine) penn-strep
) Split 1:10 in two 1Oc4 dishes with antibiotics and grown overnight at 37°C.

75λ10mM) Lis, lmM EDTA, 10μ
y plasmid DNA (omit DNA in case of mock transfection), 65λCa
C (12 and 0.65 mJ of 2X herpes buffer was carefully mixed thoroughly at room temperature and then injected directly into CHO cells with medium. Cells and DNA were incubated at 37°C for 3 hours. Then medium was aspirated and the monolayer of CHO cells was shocked with 20% glycerol-PBs (2,5+lV) for 45 seconds. The dish was flooded with 210 μL M7.8 of Fl, aspirated, and replaced with F12/DMEM. After incubating cells for 2 days at 37°C,
%XDZ.

1 by F12 medium containing FBS (extensively dialyzed fetal bovine serum) and pen-strep antibiotics.
=2 and 1:10 (split). On the fourth day, cells were fed.

On the 7th day, all the mock-transfected cells died. Transfected C according to 1:2 split
HO cells showed full growth. them, F12/
Split 1:3 in three 10 cm dishes containing DMEM 17% The dishes were fed every 4 days until a fully grown monolayer was compatible with MTX. This monolayer was then labeled with 35S-meLIXI, and secreted env fusions were measured by immunoprecipitation.

Cells diluted 1:10 in selective medium were grown at 1:50 on day 10.
It had ~100 colonies. These colonies were collected and grown in F12/DMEM. These clones were screened by radioimmunoprecipitation using anti-env antiserum.

Positive clones were amplified in increasing concentrations of MTX (500, 1000 and 3000 nM) until maximal secretion of the env fusion was observed.

Epidemiological sedimentation analysis Full-grown 10c1n dishes were prepared using RP lacking methionine.
Labeling was performed with 355 methionine at 66 pC/m in M11640 medium for 4 hours at 37°C. The medium was then collected and aprotinin (15 μL) was added.

Cells were removed by centrifugation. 1 ml of the supernatant was added to 100 μL of 2.5 M NaCl, 2% Tween (Vienna) 80.11 ng/ml BsA. 2 μL of high titer human anti-AIDS antibody was added and incubated for 1 hour at room temperature. Add protein A Sepharose to which untransfected CHO protein suzeate (lysate) has been pre-adsorbed (50 μL).
), and the resulting mixture was incubated at 37° C. for 30 minutes. Sepharose beads were then washed with PBS, 0.
It was washed three times by centrifugation in 2% deoxycholic acid, 0.2% Tween 2Q. After a final water wash, the Sepharose beads were then resuspended in SDS PAGE standard sample buffer. The sample was boiled for 5 minutes, centrifuged, and the supernatant was separated on an SDS polyacrylamide gel.

This gel was then treated with Enlighte.
n) autoradiographed at 30.

Cells producing large amounts of envelopes are anti-HTL
A ~120,000 Mr protein was synthesized that was specifically immunoprecipitated using the V-Ill retrovirus antibody method. This protein was found only in cells transfected with the plasmid.

The intracellular form of this antigen is approximately 100,000 daltons in size, and when followed by pulse-chase experiments, the secreted protein was found to be 12,000 daltons. When this protein was incubated with an anti-AIDS retrovirus, it was found that it reacted with Western blot.

D. Amplified transformation CI (cultivation of 0 cells) One 10 cm dish of total growth obtained in Step B was incubated with trypsin to release the cells, and then transferred to a small roller (rotating). 50m1 of F12 in a bottle /
DMEM50: 50 medium w/.

GHT1 low glucose, minus (-) Grinmo 7%
Transferred into XDZ PBS. CO in this bottle for 5 seconds.
2 was introduced and the lid was tightly closed. The bottle was rotated at 37°C and observed every day until full growth (~3 days).

Decant the medium and wash the cells once with PB850fflJ followed by 37 min with 5 ml of trypsin in ED'rA.
Trypsinization was performed for 5 minutes at °C. 5mg Fl
2/DMEM50: 50 moa%XDZ PBS
The resulting suspension was placed in an 850 Cm2 roller bottle. This process was repeated until the desired number of bottles was obtained. The desired number of bins is approximately 85
% full growth, add 125 g of serum-free F to each of these bottles with 4 ounces of PBS and 125 g of serum-free F.
12/DME M3; 1w10 Gf (T.

Add low glucose, -glycine and wash twice in it (
rinse). These bottles were incubated for 3 days at 37°C. On the third day, the medium was removed and kept. This process was repeated twice. ~375m1 collected from each bin
The medium was centrifuged at 2000 rpm for 5 minutes and sodium azide was added to inhibit the growth of any contaminating microorganisms.

Purification of E, l:9 recombinant HT I-V Il [Polypeptide Isolation AII] Serum of a patient diagnosed with S-related complex (/'RC) was analyzed by "Western" immunoblotting. Bankokake HT 1. , the presence of antibodies showing reactivity with V Ill protein was investigated. l-1'T
Plasma of individuals showing high titers for envelope proteins was collected and Protein A-Sepharose CL-4B was purchased from a commercial vendor [Pharmacia Fine Chemicals].
Immunoglobulins were purified by affinity chromatography using the instructions of ls)]. The substance obtained by this purification method was determined to have a purity of about 90% by subjecting it to polyacrylamide gel electrophoresis (I'ACE) and observing it by silver staining. This IM (purified) immunoglobulin was then transferred to CNBr according to the manufacturer's instructions (Pharmacia Fine Chemicals).
- Using activated Sepharose 4B, this Sepharose 4
Covalently bound to B.

The serum-free cell culture medium obtained from the fusion purification step method was harvested and 0.45 micron Nalge
n) A transparent strainer was passed through a filter and concentrated 50 times using an Amicon YM-IQ ultrafiltration membrane. This enriched medium was dialyzed against phosphate buffered saline (P 13 S ), as described in E above.

■It was applied to the affinity force ram of the type described in .

After applying the concentrated cell culture medium, the column eluate was
The column was monitored spectroscopically at 800 m and washed until the eluate was free of substances that absorb light at this wavelength. The retained proteins in this affinity column were washed with 0.1 M acetic acid and 0.5 M acetic acid.
It was eluted by treatment with NaC.5 elution buffer. spectroscopically monitoring the material eluted from the column;
Immediately treat with 1M Tris buffer to bring the pH to approximately neutral (p
Submitted to H7-8). The eluted protein was analyzed with PBS, Amicon YM-10.
It was concentrated about 5 times by ultrafiltration using Mempuran. The resulting proteins were subjected to PAGE, observed by silver staining, and analyzed by "Western" immunoblotting. Based on these analyses, the secreted, truncated recombinant I-IT
T-V IT[env fusion has a molecular weight of approximately 130 kd
was shown to be between 5 and 25 fo of the total protein eluted from the column, depending on the preparation.

Rabbits were immunized with 30-50 μg of total protein (prepared as described above) per immunization.

The primary immunization was performed by emulsifying 30 to 50 μg of protein in P 13 S 1 'me in an equal volume of complete soloin adjuvant and injecting it in the following manner: 5 ml of Q in each hind paw; and 0.2 ml sarcastic injections at 5 locations along the spine. - 14-21 days after subimmunization, rabbits were given booster immunizations. The antigen emulsion for this booster immunization was prepared similarly to that for the secondary immunization, except that incomplete Freund's adjuvant was substituted for complete Soloind's adjuvant. For booster immunization, each rabbit received two doses of the antigen, distributed as follows: Q, 5 ml on each side! intramuscularly, l
Inject me in 5 different locations along the spine (1 2ml dose of Q in each location).

Animals were bled 10-14 days after each stimulation treatment by ear vein or cardiac puncture. Antibodies raised against recombinant HTLV111 envelope protein were identified by "Western" immunoplot analysis using recombinant f(TT-V]l[130K protein as antigen. In initial experiments, 2 One of the rabbits in the wing received recombinant i (T 1., after one booster immunization).

VI [Antibodies reactive with the protein were formed. In order to induce the formation of anti-1-(T L V nf antibodies) in the other rabbit, further booster immunization was required.

Various sera from either control rabbits, rabbits inoculated with recombinant envelope antigen, normal control patients, or patients with known retrovirus-neutralizing antibodies were incubated thermally at 56°C for 30 min. was inactivated. These serum samples were then combined with it pMI 1.640.20% fetal bovine blood? %t, pen-strep, Syohi2μjq
/me was mixed with the virus at a ratio of 1:1 in a well (indentation) containing polyphrene. After incubating the wells for 1 hour at 4°C, the plate was incubated for 15 minutes.
Return to room temperature. 1 x 106 H9 human T cells were then added to each well and the wells were incubated in a CO2 incubator. On day 4, the culture was split 1=1. On day 7 or 8, cells were assayed for reverse transcriptase and subjected to immunofluorescence. Immunofluorometry uses human antisera known to contain AIDS retrovirus antibodies and is performed on fixed cells (f
ixed cell).

The proportion (%) of fluorescent cells is a measure of the proportion (%) of cells infected with the virus. Cellular host reverse transcriptase was measured as described. A decrease in reverse transcriptase levels suggests neutralization of the virus. When virus was mixed with human control serum or rabbit control serum, approximately 70-80% of the cells were fluorescent. Approximately 1.6×10 6 c pm was incorporated according to reverse transcriptase measurements.

When serum from rabbits vaccinated with recombinant envelope antigen was analyzed, the proportion of fluorescent cells was 35%;
Reverse transcriptase level was 285,000 Cpm. Human serum known to neutralize this virus has 0% fluorescence and 10.00Orp for reverse transcriptase.
m was shown. These results suggest that the antiserum obtained from rabbits vaccinated with the recombinant antigen has a neutralizing effect.

It will be understood that the foregoing describes certain preferred embodiments and that the invention is not to be so limited. It is understood that various modifications may be made to the embodiments disclosed herein, and that such modifications are intended to be within the scope of the invention.
Those skilled in the art will understand.

The citations listed in the following citations and individually cited by number in the text above are incorporated herein by reference.

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[Brief explanation of the drawing]

Figure 1 is a schematic diagram of the amino acid sequence of the amino acid/terminus of the P-24 core protein of the HTLV-Ill retrovirus, as well as the DNA sequence deduced from the amino acid sequence, and Figures 2a to 2ij are the HTLV-Ill retrovirus P-24 core protein. V-11 [schematic diagram showing the genomic DNA sequence, putative polypeptide sequence and partial restriction map; second figure is a schematic diagram of the assembly of one;
Figure 4 is a schematic diagram of expression vectors for ρ-24 and truncated P-24, and Figure 5 is assembly of expression vectors for P-24 constituent polypeptides and truncated P-24 constituent polypeptides. Schematic diagram, Figure 6 is P-DE24,
pDE24△HD3, p tl G Hp -24△I
3 and pHG Hp-24ΔHD3, Figure 7 is a replication of the Western plot of the polypeptide expressed by pI) E-24+. Figures 8a, 9a and 10a are respectively
Plasmids ptrpLE, gp41, ptrpLE, E
'Oyo iJ' P S V E , E' DF ■ F R
(7) TM diagram showing I construction, Nos. 8b, 9b and 101
) The figures show ptrpLE1gp41 and ptrp, respectively.
LE, E' and pSVE, h:' DI (FRi
A copy of polyacrylamide gel electrophoresis showing the synthesis of the encoded protein, a Pantal-copied diagram, $11a, shows HTLV-111.
A schematic diagram showing the region of the genome used for the production of env protein α.
FIG. 2 is a schematic diagram of an expression plasmid for secreting envelope proteins. Methodology 11J 1 person Genentech, Incorporated agent Patent attorney Qingbai 葆 (1 other person) Fi
g. He eight- rG GTA CAT CAG GCCB. abcdef

Claims (1)

[Scope of Claims] 1. A composition produced by recombinant culture of a prokaryotic organism or a recombinant culture of a mammal, wherein the composition is substantially free of other naturally occurring AIDS-related polypeptides. contains a predetermined polypeptide sequence of an AIDS-associated retrovirus, which does not contain any antigenic determinants that can be specifically bound by complementary antibodies. A composition characterized in that it contains one of both. 2. The composition according to item 1, wherein the predetermined polypeptide sequence is a sequence encoded by the gag region of the genome of an AIDS-associated retrovirus. 3. The composition of item 1, wherein the predetermined polypeptide sequence is derived from p-24. 4. The composition according to item 1, wherein the predetermined polypeptide sequence is a sequence encoded by the env region of the genome of an AIDS-associated retrovirus. 5. the predetermined polypeptide sequence is gp-41;
The composition according to clause 1, which is derived from gp-65 or gp-120. 6. The composition of item 1, wherein the predetermined polypeptide sequence of the AIDS-associated retrovirus, which is normally glycosylated, is non-glycosylated. 7. The composition according to item 1, wherein the predetermined polypeptide sequence contains methionine or formylmethionine at the N-terminus. 8. A method of isolating a predetermined polypeptide sequence from a prokaryotic or mammalian recombinant expression host containing a DNA sequence encoding the predetermined polypeptide sequence. wherein the isolated predetermined polypeptide sequence contains at least one antigenic determinant capable of binding to a complementary antibody. 9. A polypeptide product produced by the method described in paragraph 8. 10. A composition containing a predetermined fragment of a predetermined polypeptide sequence of an AIDS-associated retrovirus, the fragment having fewer antigenic determinants capable of specifically binding with complementary antibodies. A composition characterized in that it contains one of both. 11. The composition according to item 10, which does not substantially contain polypeptides of other AIDS-related viruses. 12. The composition of paragraph 10, wherein the polypeptide fragment is the N-terminal sequence of a predetermined polypeptide sequence of an AIDS-associated retrovirus. 13. The composition of paragraph 10, wherein the polypeptide fragment is the C-terminal sequence of a predetermined polypeptide sequence of an AIDS-associated retrovirus. 14. The composition of clause 10, wherein the polypeptide fragment is an internal polypeptide sequence of an AIDS-associated retrovirus. 15. The composition of item 10, wherein the polypeptide fragment is derived from a sequence encoded by the gag region of the genome of an AIDS-associated retrovirus. 16. The composition of paragraph 10, wherein the polypeptide fragment is derived from p-24. 17. The composition of paragraph 10, wherein the polypeptide fragment is derived from a sequence encoded by the env region of the genome of an AIDS-associated retrovirus. 18, the polypeptide fragment is gp-41, gp
-65 or gp-120. 19. A fusion polypeptide comprising: (a) a predetermined first polypeptide sequence of an AIDS-associated retrovirus having at least one antigenic determinant capable of specifically binding with a complementary antibody; and (b) a second polypeptide sequence that does not immunologically cross-react with antibodies normally present in the biologically collected sample to be analyzed for the presence of the complementary antibody. A fusion polypeptide consisting of 20. The fusion polypeptide according to item 19, wherein the predetermined polypeptide sequence is derived from a sequence encoded by the gag region of the genome of an AIDS-associated retrovirus. 21. The fusion polypeptide of paragraph 19, wherein the predetermined polypeptide sequence is derived from p-24. 22. The fusion polypeptide according to item 19, wherein the predetermined polypeptide sequence is derived from a sequence encoded by the env region of the genome of an AIDS-associated retrovirus. 23, the predetermined polypeptide sequence is gp-41
, gp-65 or gp-120. 24. The fusion polypeptide according to item 19, wherein the first polypeptide sequence and the second polypeptide sequence are fused. 25. The fusion polypeptide according to paragraph 24, wherein the fusion is by a peptide bond. 26, 19, wherein the amino terminus of the first polypeptide sequence is fused to the carboxy terminus of the second polypeptide sequence.
The fusion polypeptide described in Section. 27, a fusion polypeptide comprising: (a) a predetermined first molecule of an AIDS-associated retrovirus;
(b) for analyzing the presence of the complementary antibody; A fusion polypeptide consisting of a second polypeptide sequence that does not immunologically react with antibodies normally present in biologically collected samples. 28. The fusion polypeptide of paragraph 27, wherein said polypeptide fragment is derived from a sequence encoded by the gag region of the genome of an AIDS-associated retrovirus. 29. The fusion polypeptide of paragraph 27, wherein said polypeptide fragment is derived from p-24. 30. The fusion polypeptide according to paragraph 27, wherein the polypeptide fragment is encoded by the env region of the genome of an AIDS-associated retrovirus. 31, the polypeptide fragment is gp-24, gp
28. The fusion polypeptide of paragraph 27, which is derived from -41, gp-65 or gp-120. 32. A composition containing a mutant polypeptide sequence of an AIDS-associated retrovirus, which contains at least one antigenic determinant capable of specifically binding to a complementary antibody to the AIDS-associated retrovirus. 33. The composition according to item 32, wherein the mutant polypeptide is a fusion, deletion, or substitution of a polypeptide sequence of an AIDS-associated retrovirus. 34. The composition of paragraph 32, wherein the mutant polypeptide sequence is a labeled or conjugated derivative of a polypeptide sequence of an AIDS-associated retrovirus. 35, a diagnostic agent containing the composition according to item 1, 10 or 32. 36, a diagnostic agent containing the fusion polypeptide according to item 19 or 27. 37. A diagnostic agent containing the composition according to item 1, 10 or 32 immobilized on a solid phase. 38. A diagnostic agent containing the fusion polypeptide according to item 19 or 27 immobilized on a solid phase. 39. A diagnostic agent containing the composition according to item 1, 10 or 32, labeled with a detectable marker. 40, No. 19 or 2 labeled with a detectable marker
A diagnostic agent containing the fusion polypeptide according to item 7. 41, paragraph 1, 10 or 32, wherein the polypeptide is capable of inducing the production of neutralizing antibodies conferring resistance to infection by an AIDS-associated retrovirus. A vaccine characterized by certain things. 42, paragraph 19 or 27, wherein: (a) the antigenic determinant induces the production of neutralizing antibodies that confer resistance to infection by an AIDS-associated retrovirus; (b) the second polypeptide does not under normal conditions induce antibodies that cross-react with naturally occurring polypeptides to which the vaccine is applied; A vaccine characterized by certain things. 43. The vaccine according to paragraph 41 or 42, containing the polypeptide mixed with a pharmaceutically acceptable excipient. 44. A method of administering an AIDS vaccine, comprising administering the vaccine of paragraph 41, 42 or 43 at a dose level effective to elicit antibodies in a human subject. 45, a DNA sequence encoding a composition according to paragraph 10 or paragraph 32, which is substantially free of other naturally occurring AIDS-related DNA sequences. 46, a DNA sequence encoding a fusion according to paragraph 19 or paragraph 27, which is substantially free of other naturally occurring AIDS-related DNA sequences. 47, a replicable prokaryotic expression vector containing the DNA sequence according to paragraph 45, which when contained in a prokaryotic cell transformed by the vector,
A vector capable of expressing the composition according to item 32 or item 32. 48, a replicable prokaryotic expression vector containing the DNA sequence described in paragraph 46, which when contained in a prokaryotic cell transformed by the vector, the first
A vector capable of expressing the fusion polypeptide according to item 9 or 27. 49, a culture of prokaryotic cells containing the expression vector according to paragraph 47. 50, a culture of prokaryotic cells containing the expression vector according to paragraph 48. 51. A method for detecting antibodies contained in a biologically collected sample, the method comprising:
contact with the diagnostic agent according to any of item 0 to bind the diagnostic agent and the complementary antibody in the sample; and then b) detecting the bound or unbound diagnostic agent. Method. 52. A method for detecting antibodies contained in a biologically collected sample, comprising: a)
contacting the diagnostic agent according to any of paragraphs 1 and 2 to cause the diagnostic agent and the complementary antibody in the sample to bind; and then b) detecting the bound or unbound complementary antibody. How to become. 53. AIDS-associated retroviral reverse transcriptase that is substantially free of other AIDS-associated retroviral proteins. 54. No. 53, wherein the retrovirus is HTLV-III
Reverse transcriptase as described in Section. 55, a DNA sequence encoding the reverse transcriptase according to paragraph 53 or 54. 56, a replicable expression vector containing a DNA sequence according to paragraph 55, which is capable of expressing the reverse transcriptase according to paragraph 53 or 54 when contained in a transformed cell. A vector characterized by: 57, a eukaryotic cell culture containing the expression vector according to paragraph 56. 58. The culture according to paragraph 57, wherein the cells are of a mammalian cell line. 59, a prokaryotic cell culture containing the expression vector according to paragraph 56. 60. An assay method for identifying a compound that inhibits the reverse transcriptase of an AIDS-associated retrovirus, comprising: a) No. 57
, 58 or 59 with a compound suspected of inhibiting said reverse transcriptase; and then b) measuring the activity level of reverse transcriptase in the reaction mixture of step a). Method. 61. The measuring method according to item 60, wherein the cell culture is lysed. 62, an assay for identifying a compound that inhibits the reverse transcriptase of an AIDS-associated retrovirus, comprising: a) No. 53
or reacting a reverse transcriptase according to paragraph 54 with a compound suspected of inhibiting said reverse transcriptase; and then b) measuring the activity level of the reverse transcriptase in the reaction mixture of step a). 63. A composition comprising an E′ polypeptide of an AIDS-associated retrovirus, wherein the composition does not contain a protein derived from an AIDS-associated retrovirus-infected cell that is not encoded by the AIDS-associated retrovirus, and A composition characterized in that it does not contain virions of infectious AIDS-related retroviruses. 64. The composition of paragraph 63, wherein the E' polypeptide has a relative molecular weight by SDS PAGE of about 28,000 or about 26,500. 65. The composition of paragraph 63, wherein the E' polypeptide has the amino acid sequence of the E' polypeptide of Figure 2d, or a naturally occurring allele thereof. 66, the E′ polypeptide of FIG. 2d or a naturally occurring allele thereof, but the E′ of FIG.
A composition containing an E' polypeptide capable of immunologically cross-reacting with the polypeptide or a naturally occurring allele thereof. 67. The composition according to paragraph 63, which is a vaccine. 68, sterile and containing pharmaceutically acceptable excipients and containing an amount of E' polypeptide sufficient to elicit the formation of antibodies against the E' polypeptide when administered to an animal. 64. The composition according to item 63. 69. The composition of paragraph 63, further comprising a known amount of another predetermined AIDS-associated retroviral polypeptide. 70, a kit for a diagnostic test for AIDS-related retroviral infection, comprising the composition according to item 1 or item 63. 71. The composition of paragraph 63, wherein the E' polypeptide is labeled with a detectable group. 72. The composition according to paragraph 71, wherein the detectable group is an enzyme, a radioisotope or a fluorescent substance. 73. The test kit of paragraph 70, wherein the composition comprises an immobilized antibody capable of binding to the E' polypeptide, and the antibody is contained in a separate container from the E' polypeptide. 74, an antibody capable of binding to an AIDS-associated retroviral E' polypeptide, which does not include an antibody capable of binding to other polypeptides encoded by an AIDS-associated retrovirus, and an antibody capable of binding to an AIDS-associated retroviral E'polypeptide; Antibodies that do not contain any. 75. The antibody according to paragraph 74, which is immobilized. 76. The antibody according to item 75, which is immobilized by adsorption to polyolefin, or an antibody capable of binding to a constant region of the antibody according to item 75. 77, a method for initially determining the presence of an AIDS-associated retrovirus infection in a test subject, comprising: (a) obtaining a sample from the test subject; RN of
A method comprising detecting the presence of a polypeptide encoded by A, other than a polypeptide component of a purified AIDS-associated retrovirus virion. 78. The method of paragraph 77, wherein the detected polypeptide is an E' polypeptide. 79. The method of paragraph 77, comprising additionally detecting the presence of a predetermined polypeptide component of the AIDS-associated retrovirus virion. 80. A nucleic acid encoding an AIDS-associated retroviral E' polypeptide, which does not include surrounding proviral sequences encoding other complete AIDS-associated retroviral polypeptides. Nucleic acid. 81, a replicable vector containing the nucleic acid according to item 80. 82, No. 80 labeled with a detectable substitution
Nucleic acids described in Section.