JPH01500161A - Glycoprotein of the virus causing AIDS, method for producing the glycoprotein, and vaccine - 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] Viral vector and recombinant D encoding the glycoprotein of the virus that causes AIDS NA, cell culture infected with the vector, method for producing glycoprotein, obtained Glycoproteins, vaccines and resulting antibodies More specifically, the present invention relates to a vaccine for the prevention of AIDS. rear Innate immunodeficiency syndrome (AIDS) is currently a particular problem in North America, Europe, and Central Africa. This is a viral disease in question. According to recent estimates, 1 million Americans It has been suggested that some people may have been exposed to the AIDS virus. Patient has severe immunity The disease is generally fatal. This disease is the most common is transmitted by sexual contact. People who use intravenous drugs are also at high risk. form a group. On the other hand, by receiving contaminated blood or blood products; Many people are infected with this virus. The causative factor of this disease is a retrovirus. be. Many conditions in animals are said to be caused by retroviruses, but humans Only recently has it been possible to describe retroviruses that affect humans. .

Human T-cell retroviruses (HTLV: human T-cell leukemia virus) of type ■ and type ■ While it is believed that s) is a causative factor in some types of T-cell leukemia in adults, Lymphadenopathy, also known as type HTLV or AIDS-related virus (ARV) A related retrovirus (LAV virus) is now believed to be the causative agent of AIDS. Generally accepted.

The LAV retrovirus genome is very completely characterized (Wain- Hobson et al., 1985 HRBtner et al., 1985; et al., 1985; S. escador et al., 1985), sequence data indicate that they are closely related to the lentivirus group. Ru. Lentiviruses, derived from the sheep visnavirus, typically have long incubation periods. It is a causative factor of late-onset disease.

There are many similarities between LAV virus and Suna virus, especially in neurotropic tissue. Similar.

As with other known retroviruses, including the LAV genomic sequence, For membrane (transmembrane) envelope glycoproteins The env protein has the expected properties and is composed of gpllo and gp41. The identity of precursor gl)160 has been confirmed by direct amino acid sequencing. Below Below, gp41 is sometimes referred to as gp40 or gp41.

It corresponds to a surface antigen.

Thus, env proteins are the most promising candidates for developing vaccination strategies. Therefore, this protein and its encoding sequence are attracting attention. .

Many groups have reported the expression of macroproteins in bacteria. However, Glicoji products synthesized by these microorganisms because they are not subjected to structural modification or post-translational modification. It is possible that your immune system is weakened.

Therefore, the present invention provides glycosylation and translation vectors as env protein expression vectors. Viral vectors that allow protein expression in an environment that would allow post-structural modification I suggest using a vector.

In other words, the present invention provides a method for extracting all or part of the env gene of the virus that causes AIDS. The present invention relates to a viral vector containing the virus.

Among the available viral vectors, poxviruses are particularly important. One example is vaccinia virus (VV).

Vaccinia virus is extremely widely used around the world for the control and eradication of smallpox. It is a double-stranded DNA virus. Recent advances in technology have enabled this virus to It has become possible to develop a vector as a cloning vector, and it has become possible to develop live recombinant vectors. Viruses allow the expression of foreign antigens and a variety of viral and parasitic It is even possible to gain immunity against insect-borne diseases.

That is, several groups have recently used this type of recombinant to develop anti-influenza drugs. By expressing hepatitis B antigen and rabies glycoprotein, we develop immunity against these diseases. (Sa+ith et al., 1983, P anica li et al., 1983; Kteny et al., 1984).

It is necessary for the expression of sequences encoding foreign proteins by vaccinia virus (VV). Naturally, two steps are involved: 1) the coding sequence is linked to the promoter of vv; aligned, inserted into the non-essential section of VV DNA, and suitable bacteria plus 2) must be cloned into the middle of the coding sequence; The sequence of the VV DNA is determined by the in-vivo homology between the plasmid and the viral genome. Must allow for sexual recombination; double reciprocal recombination allows insertion D The NA fragment is transferred from the plasmid to the viral genome, where it is multiplied and expressed ( P anicali and Paolett! , 1982 HMackett et al., 1 982; Smlth et al., 1983; HPanlcali et al., 1983).

Naturally, the use of this type of vector requires It is often accompanied by partial deletion procedures.

The invention particularly relates to viral vectors containing at least: It is - part of the genome of the vector virus, -Encodes one of the glycoproteins (gp) of the envelope of the AIDS-causing virus genes, and Elements for the expression of this glycoprotein in one cell.

The invention also relates to recombinant DNA corresponding to said virus.

In the envelope of the virus that causes AIDS, their quality in KD There are three types of glycoproteins called gp160, gp120 and gp41 depending on the amount. It is appropriate to point out this. The first one, gp160 is actually is the precursor of the latter two proteins. These names are not yet established. gp41 is gp40 or gl)'! Although sometimes called 2, these 3 Despite their names, the glycoproteins of the species are considered completely identical based on their differences in mass. This is Shiuruchino.

AIDS-causing viruses include LAV virus, HTLV, Iff virus, among others. or ARV, and black mutants or partial deletions that can occur from these viruses, as well as It is understood that it refers to a virus related to.

In the part corresponding to the genome of the vector virus (different from the virus that causes AIDS) , although viral vectors may be formed from the genome of a virus of any origin. , using part of the poxvirus genome, specifically part of the vaccinia genome. It is preferable to

The conditions necessary for expression of heterologous proteins in Watashua virus are described above. .

Generally, in order for a gene in question, such as a louse gene, to be expressed, it must be It would have to be subordinate to the promoter of the cuccinia gene; The promoter will generally be the vaccinia 7.5 protein promoter. Furthermore , a non-essential gene of vaccinia whose coding sequence could serve as a marker gene. would have to be cloned into offspring. This is usually the TK gene Let's become.

Among the envelope glycoproteins whose expression we wish to achieve, the three proteins mentioned above, g Mention should be made of p160, gp'41 and gp120.

Generally, the complete envelope gene, i.e. the gene's signal sequence and Initial tests using viral vectors incorporating transmembrane sequences As a result, decoration of this gene was proposed to improve the immunogenicity of the expression product. .

Significant release of env protein into the culture supernatant was observed (this release is similar to that in vivo). probably enters the circulating body fluids). This is due to the attachment of the protein in the cell membrane. This is probably due to the fact that the antigen appears on the cell surface. It is known to be extremely important for the induction of immune responses by the senior system. follow to decorate the env protein in a manner that improves the anchoring of the protein in the cell membrane. is proposed.

To do this, replace the codon for arginine with the codon for isoleucine. To replace the transmembrane region of a transmembrane heterologous virus, e.g. For example, transmembrane region of gp of rabies virus is transmembrane of env protein. Possibilities that may improve the anchoring by substituting and/or adding to the membrane region There is also gender.

Furthermore, the protein may not be fully assembled after its expression. In practice, Gnar peptides are highly atypical and may impair complete export of the protein. Ru. For this reason, signal sequences derived from foreign viruses, such as rabies virus It is proposed to replace and/or add the signal sequence of gp.

Finally, it appears that gp120 rather than gp160 is released by cells. It seems like that. On the one hand, it provides a decoy for the immune system, and on the other hand, recently data, it would be able to bind to T4 cells. This combination is The effect of inactivating T4 cells or making them appear foreign to other T4 cells It would have the effect of

Therefore, it would be advantageous to obtain gpl20env proteins that cannot be released. . This combines the env gene with sequences encoding gp120 and gp41. a protease located between gp120 and gp41. By removing the cleavage site, in particular by removing the REKR site, achieved.

In the plasmid of the present invention, the KRR sequence is located 8 amino acids downstream from the REKR sequence. At least one additional mutation is made at the site corresponding to the column. That's how you get it gp160 is no longer cleaved.

The vector of the present invention further improves the ability of the env protein to form a syncytium. is responsible for the ability of viruses to cause cell fusion and produce giant cells or syncytia. Corresponds to the hydrophobic N-terminal peptide of gp41, which is thought to be present. Contains the sequence encoding gp'41, which lacks sequence.

Finally, the present invention provides that the extracytoplasmic region and the intracytoplasmic region of gp160 have a C-terminal hydrophobicity. Secretion of the glycoprotein to be synchronously fused after peptide deletion and thereby obtained This article concerns the viral vectors that make this possible.

Generally, the viral vectors of the invention contain sequences encoding one of the following proteins: Do it -S-gp120yogp40- (tm)-・S is a signal peptide, ・gp120 is glycoprotein 120, ・J is gl) between 120 and gp40. A conceptual representation of a junction lacking a protease cleavage site; ・gp40 is glycoprotein 40, -tIll is a transmembrane peptide.

or -3gp40-tm- Or -8-gpl 20-tm- A protein with the following structure.

The signal peptide and transmembrane sequences are those of the AIDS-causing virus. even if it is of a different species (especially rabies virus or vSv or E). They may also be derived from other viruses with envelopes.

gp40 may lack its hydrophobic N-terminus.

The first invention mainly focuses on the glycoprotein encoded by the env gene of the LAV virus. It concerns the use of viral vectors to obtain white matter in cell culture. follow First, the cells are mammalian cells infected with the viral vector of the present invention. , or mammalian cells containing the corresponding recombinant DNA; In particular, we will mention human diploid cells from primary culture and Vero cells. should be. Naturally, as can be seen from the examples below, other types of It is also possible to provide cells.

The glycoprotein thus obtained can be used for vaccine production after purification.

It is also possible to use the viral vectors of the invention directly for vaccination; When the glycoprotein is produced at the required site and within the body.

Two or more types of immunizing agents, especially gp120 and gp40 attached to the above decoration, individually Using a combination of immunizing agents corresponding to vectors expressed in It is advantageous to give For example, derived from vectors 1136 and 1138 described below. It may be advantageous to use combined vaccination agents.

Finally, the present invention also relates to antibodies produced against the above-mentioned glycoproteins. , the antibody was induced after a certain period of time after infecting a living body with the above viral vector. Obtained by collecting antibodies.

The techniques used for glycoprotein acquisition, cell culture and vaccination are consistent with known vaccines. This is the same as what is currently being done, so I will not discuss it in detail.

The invention will be better understood from the following methods and examples.

The five figures illustrate the examples: Figure 1 shows recombinant VVTGeLAV 9-1 and VVTGeLAV1132, and was immunized using anti-LAV serum. The effect of endo-F on precipitated proteins is shown. In this diagram, Molecular weights are given in kilodaltons and have the following signs: ・P1 cell Beret, ・S1 skim, ・Product obtained without u1 treatment, -e, product obtained after endo-F treatment.

Figure 2 shows that LA was treated with serum from mice inoculated with recombinant VVTGeLAV9-1. This shows how V virus proteins are recognized. In this figure, T is the serum used. Indicates when the patient is suffering from AIDS. Molecular weight is expressed in kilodaltons. Figure 3 shows the immunoprecipitation of the protein. In this diagram, molecular weight is expressed in kilodaltons. It is shown.

Figure 4 shows recombinant viruses vv, TG, eLAv1135.1136.1137 1138 and 1138 are shown. Virus 1135 is cultured It synthesizes gp160, which does not appear in the culture supernatant. Viruses 1136 and 113 8, respectively, associated proteins gp120 and gp40 associated with cell pellets. produce. Virus 1137 produces a slightly smaller protein than virus 1135. The molecular weight corresponds to that expected.

Figure 5 shows the structure of the env protein synthesized by the recombinant virus described in the present invention. It shows the structure.

S: Signal peptide H: internal hydrophobic region TM Nidorans membrane mooring area ↑: gpl20/gp40 cleavage site lI: Sequence derived from rabies glycoprotein Enzymes: Use according to supplier's instructions.

The only difference: Human 143B cells were used instead of LMTK+ cells.

Preparation of preserved virus "Sterile" chicken primary cells were grown at 0.01 pfu/cell for 4 days at 37°C. Infect with (MEM medium + 5% NC (rotor GSA, 5orval) l) o Keep the supernatant.

The pellet was washed with RSB buffer (10mM Trls-HCQ, pH 7, 4, 10mM Treat in KCQ, 1mM MHCO3) for 15 minutes at 4°C. Ceramic container for suspension (Potter) and centrifuged at 250 rpm for 15 minutes.

Add the supernatant to the above-mentioned supernatant and perform the second mashing in the same manner.

All supernatants were transferred to a 36% (W/V) sucrose cushion (1,0mM Tri s, pH 8) Place on top of 10mQ. The suspension was centrifuged for 2 hours at 14.00 rpm. Isolate the heart (Rotor 5W28, Beckman).

Take out the beret, crush it and place it on another identical cushion. Second beret into PBSSmQ and a 20-40% sucrose gradient (10mM, Tris 5pH8) (same rotor). Suspension at 12.00 rpm Centrifuge for minutes.

Collect the virus band. Centrifuge it at 20,000 rpm for 1 hour. Make it into a beret. Take up the pellet in 10mM Tris, pH 8.

immunoprecipitation Infect BHK-21 cells with 0.2 pfu/cell for 18 hours (def. Dish diameter 3 cm s cell number 106/dish, G-MEM + 10% FC 8). Decant the medium and add one time to methionine-free medium and [35S]methionine. Replace with Onin (Amersham) 10 u Q/dish.

After 2 hours, add excess cold methionine.

After labeling, infected cells were scraped, centrifuged for 1 min in an Eppendorf centrifuge, and supernatant. The clear and pellet fractions were separated, the pellet was washed once with PBS buffer, and then immunized. Perform sedimentation and perform gel electrophoresis (following L.Athe et al., 1980) .

Endo-F treatment After immunoprecipitating the labeled protein with AIDS patient serum, protein A-Sepharose fractionation was performed. minutes 0.2M sodium phosphate, pH 6.1 0.05% 5DS 0.1% Nonidet P40 0.1% β-mercaptoethanol 0.1% EDTA p)(s Boil for 5 minutes to denature the protein.

111112 with 4 units of endo-F at 37°C for 20 hours. tion followed by precipitation with 115 volumes of 100% TCA in water for 2 minutes. . Wash the pellet three times with 80% acetone, add sample buffer, and transfer the mixture to an SDS gel. E, using a sheep anti-mouse antibody linked to peroxidase as a second antibody, Adopted LAVIA test (Pasteur-D iagnostic).

vaccinia A 96 flat bottom well plate (NUNC) was prepared with 107 pfu of wild type vaccinia in carbonate buffer. Incubate with virus for 18 hours at 37°C. Next, the plate is 0.01 % gelatin. Mouse serum was then adsorbed onto the plate, and the remaining operations were carried out. The production is performed in the same way as for LAV ELISA.

Read at 492nm.

The total size of the various elements necessary for its later expression is on the order of several kb. Therefore , to facilitate the necessary manipulations, a platform for replication in E. coli used for construction work is required. It was considered necessary to reduce the size of the sumid to an extremely small size.

The H1ndm fragment (Hln-J) of the VV genome is used for chitidine kinase (TK). Contains the complete gene for the vv genome, which contains the replacement DNA inserted into the vv genome. and has already been used previously for recombination (Mackett et al., 1982) .

Transfer of the insert fragment into the TK gene of the vv genome creates a selectable TKK-defective virus. It is important to mention what happens. First, VVHln-J fragment Generate a small plasmid with a single H1nd■ site that can be used to integrate It was necessary. In addition, unnecessary restrictions have been removed to allow the following operations: It was necessary to remove the column from the plasmid.

The construction was carried out using a vector derived from plasmid pBR322 by natural deletion. Plasmid p in which the segment between leotides 1089 and 2491 is missing Starting from ML 2 (Lusky and Botchan, 1981). destination First, the "linker-tiling" method (Lathe et al., 1984) was used to remove the linker.

This results in a functional β-lactamase gene (which confers ampicillin resistance). and a replication origin active in E. coli and a single H1ndIII restriction site. A 2049 base pair plasmid containing

This construct is called pTGIH.

The Hln-J fragment of VV DNA containing the TK gene was first extracted from pBR327. cloned into a vector (Drillien and S. pehner, (1983). This 4.6kb fragment was re-clipped into the HindIII site of pTGIH. I turned it into a loan.

A clone in which the TK gene is located distal to the gene encoding ampicillin resistance. selected.

This construct pTGIH-TK was used as a vector in the following experiments.

The next step is to control the expression of the sequence encoding the gene to be expressed. The obtained vv promoter was isolated. 7500 Daltons (7,5K) The promoter of the early gene encoding the protein has already been successfully used for the same purpose. (S with et al., 1983).

Therefore, an attempt was made to isolate this segment.

(V enkatasan et al., 1981). Smaller pieces are preferentially crawled. The 5alI cut WR type vv DNA is inserted into plasmid pBR322. directly cloned into cteriophage M 13mp701 (Kieny et al. , 1983). This yielded phage M13TGSal-8. Ru.

In this clone, the end immediately after the starting ATG of the gene was inserted into the E. coli clone at 7.5. Fill with no fragments and then shift. At the same time, downstream 5alI (Acc I) Site removed This construct is called M137G7.5.

(1983). M13TG7.5 is cloned into 7,5 present in pTGI Generates H-TK-P7.5K.

Use the part for the next construction.

Bacteriophage Ml 3TG 131 (Kieny et al., 1983) The relinker segment was excised using EcoRI and BglII, and the plasmid was Insert between EcoRI site and BamHI site in pTGIH-TK-P7.5. to generate pTG7186-POLY. This construct has controls on P7.5. There are 10 restriction sites available for cloning your foreign genes.

To obtain sequences encoding env, plasmids PJI9-6 and PJ19- The two provirus segments cloned into 13 are first allowed to associate.

The putative translation start site of the env gene to ensure that the env mRNA is fully translated. Match the nucleotide sequence surrounding the position to the consensus sequence of eukaryotic genes. It was decorated to look like. This was done using an oligonucleotide proximal to position 5767. This was achieved by directed specific mutagenesis.

Plasmids PJ19-13 and PJ19-6 each contain nucleotide 1258 ~1698 and 1698-917G) into phage M13TG130. and oligonucleotide (sequence 5'-CTCTCATTGTCACTGCAG TCTGCTCTTTTC) to directionally and specifically insert the mutated fragment into plasmid pTG. 1-POLY (2.lkb mini-plasmid similar to pTGIH, but M 13TPJ19-13-derived Tamaμro Bindu fragment into the same plasmid as between KpnI and H1nd■), followed by Hl of PJ19-6. ndm-XhoI fragment was cloned (Hlndm and 5alI ), giving rise to the complete env coding sequence sandwiched between two PstI sites. (plasmid pTG1124).

By introducing these two PstI restriction sites, env Manipulation of genetic DNA becomes easier.

As mentioned above, expression of a heterologous protein with vaccinia virus requires that the coding sequence Aligns with the senior promoter sequence and contains non-essential (dispensable) segments of vaccinia DNA. be inserted into the client. This DNA located on both sides undergoes double reciprocal recombination. This makes it possible to recombine with the vaccinia genome in vivo, thereby The code sequence and associated promoter are transferred into the vaccinia genome.

For this purpose, the above PstI-PstI fragment was added to the PstI-PstI fragment of pTG186-POLY. Cloned into site I. This creates a plasmid called pTG1125. Obtained.

Plasmid pTG186-POLY is derived from plasmid p'rG188 by PstI It can be produced by digestion and religation with T4 ligase.

Plasmid pT0188 was introduced on June 20, 1985 into the French country 75015 virus. Riichi Doctor Wool Ru28 Nokustool Research Institute's National Microbial Culture Collection It has been deposited with the following number: E. coli 5KpTG 188=Na, I458env gene coding sequence and the accompanying promoter into the vaccinia genome is accomplished as follows. .

The site method described by Sm1th et al. (1983) inserts into the VVTK gene. By causing in vivo exchange between the plasmid containing the fragment and the wild-type virus genome, It is based on inactivating the TK gene of the virus. TK”Will Cell lines (TK-deficient ) can be selected by brating (Macket et al., 19 1982). Thymidine kinase phosphorylates 5BUDR to monophosphate. . This is then converted to triphosphate. This compound is an analog of dTTP and its incorporation into DNA inhibits the proper growth of the virus.

However, the TK+ virus replicates its DNA normally and is still transferred to the TK+ cell line. and produce visible viral plaques.

Vaccinia virus multiplies in the cytoplasm of infected cells rather than in the nucleus. For this reason , unable to utilize the host DNA replication and transcription machinery, resulting in virions (viruses) It is necessary for the microorganism particle to have various elements for its genome expression.

Purified VV DNA is non-infectious.

Generation of recombinants involves infection of cells with vV virions and cloning of the cloned subject. Transfection and transfection with DNA segments must be performed simultaneously. deer However, the generation of recombinants is competent for transfection with DNA. limited to a small percentage of cells. Therefore, from a non-recombinant parent virus, Indirect “congruence” to reduce background It was necessary to adopt the J method. As a live infectious virus, this A temperature-sensitive (ts) vaccinia species that cannot grow at the non-permissive temperature of 9.5°C. This was achieved using the nom (Drillfen and 5pehner, 1983). . Cells were infected with the ts mutant strain under nonpermissive conditions and challenged with wild-type virus DNA. When transfected, the cells are competent and wild-type for transfection. Only in cells where recombination between the virus DNA and the ts virus genome has occurred, the virus virus multiplication will occur; in other cells, even if they are infected, will not proliferate. Containing vaccinia DNA fragments, such as pTG1125. Recombinant plasmid with wild type at appropriate concentration in the transfection mixture It is also possible for Vaccinia DNA to participate in homologous recombination with vaccinia DNA in a symbiotic relationship. Ru.

Monolayers of primary chicken embryo fibroblasts (CEF) were incubated with vv-Copenha at 33°C. The wild type Vv-Copenha Gene virus DNA (50 ng/106 cells) and recombinant plasmid (50 ng/106 cells) transfected with calcium phosphate co-precipitate with ng/106 cells).

Incubate for 2 hours at a temperature (39.5°C) that does not allow the growth of ts virus. After this step, the cells are incubated again at 39.5° C. for 48 hours. ts+ Reinfect monolayers of human 143B cells at 37°C with diluted virus. , and then incubated the cells in the presence of 5BUDR (150 μg/body). Ru. A plug of TK+ virus is seeded from these cells that have received the recombinant plasmid. The control cultures without plasmid show no visible plaques. T The K+ virus is then subquenched by a second round of selection in the presence of 5BUDR. Rowing.

Correct double interaction between hybrid plasmid pTG1125 and vv genome When the replacement occurs, the TK gene containing the inserted fragment intersects with the TK gene of the virus. The recombinant becomes TK+.

DNA purified from various rK+g recombinant viruses was digested with H1nd■. Subject to low gel electrophoresis.

The DNA fragments were described by 5outhern (1975) 1. Follow the method and transfer to a nitrocellulose filter. Next, the filter was filtered using 32P. Hybridize with plasmid pTG1125 after translation.

After washing the filter, fluorography is performed to detect vaccinia virus. If the env gene of V is integrated, 3.85-1 will appear on autoradiograph 7. 2.9- and 0.8-kb bands are visible. One of these recombinants, VV , TG, and eLAV9-4 were selected for the following experiments.

To prove gene expression, G-MEM medium + 1 width recombinant VV-TG, Infect eLAV9-1.

Infect fresh semi-confluent monolayers (106 cells) with 0.2 pfu/cell. and incubate for 18 hours.

The medium was then removed and a low methionine solution containing 10 μg/IQ of [35S] methionine was added. Add thionin-concentrated medium (1 drop for 106 cells). Ink cells at 37°C. The labeled proteins are collected by centrifugation. For beret and skim After separation, the proteins are incubated with serum from AIDS patients. The serum and Reacting proteins were collected by adsorption to protein A-Sepharose resin, and SDS Spread by polyacrylamide gel electrophoresis and perform autoradiography ( (according to the method of L.athe et al., 1980). The autoradiograph is We show that serum from patients with cancer specifically binds to three types of proteins in infected cell extracts. (The results are the same or similar to those obtained using serum from other patients).

Apparent molecular weights of 160, 120, and 41 KD were found in standard env glycoprotein preparations. g, which was confirmed using serum from AIDS patients in extracts of LAV virus-infected cells. Suggests equivalence with the p160, gp120 and gp41 bands. L.A.V. Three types of proteins are expressed from a recombinant vector containing only the coding sequence of the env gene. This observation may be due to proteolytic cleavage of the -translated product gp160. This supports the hypothesis that gp120 and gl)41 are generated.

The lice encoding sequence results in a primary translation product of approximately 90 KD. On the other hand, the above method The env precursor obtained in the above has an apparent molecular weight of about 160 KD. This difference is Attributable to the presence of a significant amount of glycosylation. Ene to remove glycosyl group The expected product obtained according to the invention by digestion with doglycosidase F A good correlation with the product could be demonstrated (Figure 1).

VV, TG, eLAV9-1 was administered to 7 male Ba1b/e mice at 2 weeks of age at 5 doses per animal. Inoculate by subcutaneous injection with 07 pfu of X1.

Two weeks later, the mice received a booster injection at the same dose and received 1, Blood samples are taken after 2 and 4 weeks. The LAV virus was present in their serum. The presence of antibodies against antigenic determinants of the Vaccinia virus and Vaccinia virus is examined.

All inoculated animals had blood that could react with vaccinia virus in the ELI SA test. give cleanliness. In contrast, the response in the EL ISA test against LAV virus is weak and has low reproducibility. In order to improve the sensitivity of the test, We used the "Ning" method. This method uses antibodies that can react with LAV virus proteins. The proteins were denatured with SDS in an electrophoresis gel and transferred to a nitrocellulose membrane. Make it possible to prove it after moving it to In this experiment, the nitrocellulose used The film is LAV sold by Diagnostic-Pasteur. -It is that of BLOT, to which the LAV virus protein has already been bound. Ru. These membranes were cut into strips, and each strip was inoculated with mouse serum (1/20 dilution). Incubate with. Second antibody conjugated to peroxidase (sheep anti- mouse) allows visualization of LAV viral proteins bound to mouse antibodies .

Some sera (12/27) had a molecular weight of approximately 16, which corresponds to gp160 of env. It exhibits a specific reaction with 0KD protein (Figure 2). In some sera, gp4 1 protein is also observed. A small number of mouse sera were collected on Western blotti generation of a signal corresponding to an unidentified protein of the membrane-bound LAV virus. Please note that (this plasmid pTG1128 should be The sequence encoding the Blen region has been mutated, replacing arginine with isoleucine. It is the same as plasmid 1125, except that it is replaced by this The mutations are intended to improve the attachment of the protein in the cell membrane.

insert. This yields phage M13TG154.

Next, for this phage M137G154, the codon encoding arginine was Localized mutations designed to replace codons encoding isoleucine Perform the trigger.

For this purpose, the following oligonucleotide is used: 5' GGTTTAATA ATAGTTTT3' Thus, phage M13TG155 was obtained, whose sequence is modified as follows: Gly Leu Arg Ile Val Original arrangement: GGT TTA AG A ATA GTT mutation sequence: GGT TTA ATA ATA GTTie The thus mutated BamHI-Hlndm fragment was extracted from M13 TG155. Plasmid pTG1127 was transferred into the corresponding site of lasmid pTG1124. obtained, in which the arginine codon is replaced by an isoleucine codon. The PStI-NizenI fragment of pTG1127 was plated as described in Example 1. The plasmid was cloned into the PstI site of Sumid pTG186-POLY to pTG1128 is obtained.

This plasmid contains a sequence encoding the transmembrane region of the rabies glycoprotein. The sequence is fused to the beginning of the hydrophobic protein-encoding sequence of the env glycoprotein.

The transmembrane region of the rabies glycoprotein is present in the phage, thus phage M1. 37G156 is obtained.

Next, we performed localized mutagenesis on M13TG156 to identify the env sequence and rabies The sequence was converted to 5'GCTGTGGTATATAAAA using oligonucleotides. synchronously by forming a TATGTATTACTGAGTG3' loop. fuse.

Tyr Leu Lys lie Phe Gly Lys Tyr, Val? Thus, phage M13TG157 is obtained.

Rabies glycoprotein transmembrane (tm) that has just been fused with the env gene. ) region is then transferred into plasmid pTG1124.

■ parts are broken).

The BglII site of M13TG157 is a rabies gp fragment: Balt! BglII Plasmid pTG1126 is thus obtained.

Obtain plasmid pTG1130.

This method involves fusing the signal sequence of the rabies glycoprotein with the signal sequence of the rabies glycoprotein.

The signal sequence of the rabies glycoprotein was transferred from the plasmid pTG115PRO to BglII. -Hind was extracted in the form of a m fragment and using a single-stranded adapter with the following sequence. Clone into the PstI-Hlndm site of M13TG130: 5' GATCTGCA3' Thus, phage M13 TG158 is obtained.

Next, the env signal peptide was transferred to M137G158, and this It is fused with the gene encoding the signal peptide of the rabies glycoprotein.

For this purpose, KpnI was used with S1 nuclease followed by Klenow and EcOR enzymes. Pst”/HLndIr Engineering”Pst”/BglBglII Null Signal <-111(- Thus, phage M13 TG159 is obtained.

Transfer into TG131 to obtain plasmid M13 TG160.

When performing localized mutagenesis on M13 TG160, the env sequence and rabies glycoprotein White matter sequences can be synchronously fused (forming loops). This is the following oligo Achieved using nucleotides: S’ GACCCACAATTTTTCTGTAATAGGGAATTTCCC λAA3″ Rabies signal === ■Big Mouth Gly Lys Pha Pro Ile Tyr Thr Gi Thus, phage M13 TG161 is obtained.

do). This yields plasmid pTG1129.

Plasmid pTG was created by cloning into lasmid pTG186-POLY. Get 1131.

Mido M13 TG162 is obtained.

Then the oligonucleotide 5’　ATTCCCACTGCTTAGTATCATTCTGCACCAC Limit mutagenesis is performed using TC3'.

This allows the stop codon to be located at the end of gp120. obtained The resulting array is as follows: N　E　Y　**x Mutation sequence: , CAG AAT GAA TACTAA GCA GTG thus Obtain phage M13 TG168.

MidopTG1132 is obtained.

Example 10 By localized mutagenesis of M13 7G162 using the following oligonucleotides, Bacterial phages in which the cryptic cleavage site separating gp120 and gp40 have been destroyed. Get page: 5' ATTCCACTGCTTGGTGTTCATTCTGC The ACCACTC3' decorated sequence is as follows.

Cutting site Mutation sequence: CAG AA'Z' GAA CACCAA GCAEHQ Thus, phage M13 7G165 is obtained.

Obtain plasmid pTG1133.

DiM13 TG163 is obtained.

To destroy the same gl)120 cleavage site as above, to perform localized mutagenesis. The following oligonucleotides are used for this purpose.

5' ATTCCACTGCTTGATGTTCATTCTGACCACT C3' This allows us to decorate the array as follows: EKR Original sequence -G AGA GAA AAA AGAiGCA GTG These Under the conditions, phage M13 TG166 is obtained.

As a result, plasmid pTG1134 is obtained.

Working as described above for plasmid pTG1125, prepared above Obtain hybrid vaccinia vectors compatible with various plasmids.

Each of these viral vectors VV, TG, eLAV1128 VV, TG, eLAV1130 VV, TG, eLAV1131 VV, TG, eLAV1132 VV, TG, eLAV1133 VV, TG, eLAV1134- Name it.

The protein obtained as described above is tested by immunoprecipitation (Figure 3).

For virus 9-1, the immunoprecipitate groups were gp160, gl)120 and gp Immunoprecipitation corresponding to 41 is shown.

The same applies to virus 1128.

Virus 1130 also exhibits gp160 and gp120.

The protein corresponding to gp41 has a slightly lower molecular weight due to its C-terminal decoration.

Virus 1131 has virtually identical specs to those obtained with virus 1130. Toll is shown.

Virus 1132 naturally does not show a protein corresponding to gp41. Pe The 105KD protein present in RET is an isoform of gp120 (glycodiform). (differences in standardization).

For virus 1133, this clearly shows proteins 160, 120 and 41. However, the bands corresponding to proteins 120 and 41 are weaker than in other spectra. stomach.

The same is true for virus 1134, where 41(7) molecules: jl< Low squid, virus VV, cleavage compared to TG-1125 (9-1) ~ 1131 It is clear that this is happening at a slow rate.

Kinetic analysis of release performed on VV, TG-eLAV1133 and 1134 are gp120 and gp'! Although the speed of disconnection between 1 and 1 is slow, disconnection still occurs. to show that Examination of the DNA sequence of the env gene reveals that there are 8 nucleotides from the first cleavage site. It is found that there is another potential cleavage site (KRR) downstream of the amino acid. Therefore, This second site is used to obtain a recombinant vaccinia virus that expresses only gp160. It would be important to mutate the .

By localized mutagenesis of M137G166 with the following nucleotides, a second Obtain phages decorated with cleavage sites: 5’　ATTCTGCACCACGTGATTCTGTGCCTTGGTηG The T3' decorated sequence is: Original sequence *GCA AAG AGA AGA GTG mutant sequence t GCA GAG AAT CACGTGQNHV The obtained phage (M13 TG181) was ligated with PstI and Generate mido pTG1135.

en synthesized by recombinant vaccinia vector VV, TG, eLAV1135 The C-terminal part of the v gene is a sequence derived from rabies glycoprotein. Therefore, this C-terminal Another recombination in which the C-terminal part of the env gene of the LAV virus was replaced. Having a body also seems useful.

For this purpose, phage M13 TG166 (see Example 13) and The same mutagenesis was performed on phage Ml 370165 and phage M13 gives rise to TG184.

The PstI-PstI fragment of M13TG184 was then transformed into plasmid pTG186-P. Cloned into OLY to generate plasmid pTG1139.

It is also desirable to have a recombinant vaccinia virus that expresses only gp120. Dew. This gp120 is VV, TG.

Unlike the one obtained with eLAV1132 virus, the C-terminal anchorage region will be equipped with.

For this purpose, localized mutagenesis using the following nucleotides, 7y-diM13 TG181) gp401:l: Remove the corresponding sequence: 5'TGCACTCAGTAATACATACACGTGATTCTGTGCC τT 31 This oligonucleotide contains the gp12o sequence (decorated cleavage site ) and the sequence of the transmembrane region of the rabies glycoprotein. allow for fusion.

KAQNHVV FYVLL Thus, phage M137G182 is obtained. Confirmation of phage M13TG182 The Shakutan I fragment was then inserted into the PstI site of pTG186-POLY to create a plasmid. gives rise to pTG1136.

Little is known about the role of the hydrophobic region located at the N-terminus of gp40. giant protein This region of quality may be responsible for the ability to induce syncytium formation.

Therefore, it may be advantageous to generate gp160 that does not have this sequence. Ru.

For this purpose, we encoded this hydrophobic peptide in phage M13TG181. Fuse the upstream and downstream sequences of the part using the following oligonucleotides. TTGT (τGGCCτGCACGTGAT”rCTGT GCCTT 3' This can be obtained from phage M137G18 by performing the following fusion. Make it possible to obtain 3: Enter AG GCA GAG AAT CACGTG GTG -)-GTA CA G　GCCAGA　C Input M13TG183 PstI-PstI fragment into pTG 186-POLY into the PstI site to create plasmid pTG11. It gives rise to 37.

In addition to recombinant viruses expressing gp160 or gp120, gp40-only It would be useful to generate recombinant vaccinia viruses that express

For this purpose, the sequence encoding the signal peptide was added to phage M137G163 Fuse with the gp40 encoding sequence above using the following nucleotides: 5° CAA'rAATTGTCTGGCCTGAATAGGGAATTTCCCAA A3' This would allow the generation of phage M13TG180 containing the next fusion. Melt: τGT 'rTT GGG λ input τTC-#-GAG GCCAGk Cλ Signal of rabies GP GP40 screaming part 3TG180 bile knee shakudan The fragment was inserted into the PstI site of pTG186-POLY to create plasmid pTG1. Get 138.

As in the case of gp160 (plasmid pTG1139), the anchoring region and The intracytoplasmic region does not correspond to the sequence of the rabies glycoprotein, but rather the env of the LAV virus. It would also be important to have a recombinant virus expressing gp40, a gene sequence.

phage M13TG190.

By cloning into the PstI site of Sumid pTG186-POLY, Obtain plasmid pTG1162.

Recombinant vaccinia virus that synthesizes gp160 that is secreted into the medium without being cleaved It also seems important to obtain support. In reality, this protein is used as a killed vaccine. , in combination with an adjuvant or liposomes or IS COM S CMorei n et al., Natire (1984) 308.5958, p457-603. You can put it in and use it.

For this purpose, sequences encoding the extracytoplasmic and intracytoplasmic regions are Bacteriophage M13TG fused synchronously in M13TG184 194 is constructed using the following oligonucleotides: 1163 is obtained.

The recombinant protein thus obtained, in particular non-cleavable gp160, is It can be used in diagnostic kits to detect potential antibodies present in a patient's blood.

These tests can be carried out using methods well known to those skilled in the art, such as ELISA, RIPA or Can be performed by “Western blotting” (immune imprinting) .

These proteins were designed to detect the presence of the virus in a sample. It can also be used for the production of monoclonal antibodies and hybridomas.

The various plasmids and M13 phages used are described in the following patent applications, etc. Listed: M13 TG131: Kieny et al., 1983 M13 TGR G151: WO33104052pTG155PRO: FR8406499M 13TG130: Kieny et al., 1983 The following plasmids were introduced in 1984. On November 16th, it was added to the National Collection of Microbial Cultures (CNCM) at the Institut Pasteur. Deposited and described in patent specification GB-A-84/29.099 : PJ19-6: CN0MNα366-IPJ19-13: CNCM No. 367-I plasmid pTG1125 was collected from the same collection on June 6, 1986. In addition, transformed bacteria E, coli 106/pTG1125, N o, l-557.

Reference materials 1. Drillien, R., Spener.

D + (Spehner, D,), Virology, 131, 385-393 (1983) 2, Keeny, M, P, (Kieny, M, P,), Rase, R.

(Lathe, R,), Lecocq, J, P, (Lecocq, J, P,) .

New Earth Tile Cloning and Sea Fencing Vector Zu Beist on Bacteriophage M 13 (New versati cloning and sequencing vectors base ed on bacterialphage M2S), Gene, 26.91-99 (1983) 3, Keeny, M. , P.), Rasse, R.

(Lathe, R,), Drillien, R, (Drillien, R,), Spehner, D., Scoley, Varnish.

(Skory, S.), Schmitt, D. (), Victor Wiktor, T.: F Browski, H.

(Koprowski, H,) + Lecoc, Jay, P, (Lecoc q, J.

P, ), Expression on Labez Virus Glycoprotein Flo Recombinant vaccinia virus (Expression of rabies vlrus glycoprotein from a rec ombinant virus), Nature ), 312, 163-166 (L984) 4, Lathe, R, ), Haas, P ().

Dewilde, M., Half-Ord, F.

(Harford, N.), Lecocq, J. P.

), Cell-Free Synthesis on Biological Active Heat Stable Enterotoxin On + 1 From A Clone Dosie Cell-free 5 synthesis of biological y active heat-stable enterotoxin of’ colt from cloned gene), Nature, 284 ．． 473-474 (1980) 5, Lathe, R8, Keeney, em, p.

(Kieny, M.P.), Schmitt, D. (), Kerr. Chis, Bee (Curtis, P.), Rukotsuk, Ji, Bee.

(Lecocq, J, P,), J, Mo1. Appl, Genet, , 2.331-3426, Lathe, R., Keeney , M, P.

(Kieny, M.P.), Scots. Varnish, (Skory, S.), Lukotsk .

J, B, (Lecocq, J, P,), DFA (DNA) , 3, 173-182 (1984) 7, Lusky, M. M.), Boccan, M.

(Botchan, M.), Nature, 293.79-81 (198 1) 8, Mackett, M., Smith, G., L.

(Smith, G, L,), Moss, P, (Moss, B,), Vaccine Avirus: A selectable eucario take cloning and Expression Vector (Vaccinia virus: a 5el ectale eukalyotic cloningand express ion vector), Proceedings on the National Academy -On Science On The United States On America (P roc.

Natl, Acad, Set, USA), 79.7415-7419 (1982) 9, Maniatis, T. Lish.

E, F. (Fritsch, E. P.), Samprutsk, J.

(Sambrook, J.), Molecular Cloning Near Laboratory -Manual (Molecular atoning: alaborator y manual), Cold Spring Harbor Laboratory, New York (Cold Spring Harbor Lab.

,N,Y,) 10, Muesing, M, A, (Muesing, M, A,) * Smith , D, H, (Smith, D, H,), Cabradella, C.

D, (Cabradilla, C, D,), Benton, C, V.

(Benton, C, V,), Lasky, L, A, (Lasky, L ,A,).

Capon, D, J, (Capon, D, J,), Nucleitsquacid Struggle and Expression on the Human Aids / Lymph denovasy retrovirus (Nucleic acid 5 truct ure and expression of human AIDS/lymp badenopathyretrovirus), Nature, 313 , 450-458 (1985) 11, Messing, Sai Vieras, Gene.

19.289-276 (19g2) 12. Pnicall, D., Baoletti, E.

(Poletti, E.), Construction on Box Virus Illustrator Zu as Cloning Vectors: Insertion ° on the Thymidine Kinase gene funia virus (Construction of po xvirus as cloning vectors: 1nsertion o f the thymidlnekinase geneform herp es simplex virus 1nto the DNA offinfe ctious vaseinla virus), Proc, Natl, Acad, Sci.

USA, 79.4927-4931 (1982) 13, Vanikari, D. (Pan1cali, D,), Davis, Nis.

Davls, S.J., Weinberg, R., L.

(Welnberg, R, L,), Paoletti, E. (Polettl ,E,).

Proc, Natl, Acad, Sci, LISA, 80.5384 -5388 (1983) 14, Ratner, L. l　Haseltine, W, (Haseltlne, V,), butter force, R , (Patarca, R.).

Llvak, K. J., Starkich, B. + (Stareiehi, B.), Josephus, Nis, F.

(Josephs, S.F.), Tran, E.R., (Doran, E. .

R1), Rafalski, J, A, (Rafalski, J, A,).

Whitehorn, E, A, (Vhitehorn, E, A,), Baumai Star, Kay, (Baun+eister, Ni,), Ivanov, El.

(lvanoff, L.), Petterway Jr., Niss, R.

(Petterway Jr., S, R,), Pea son, M.L.), Reutenberger, J.A.

(Lautenberger, J.A.), Babasu, Ti, Varnish, (Pap as, T.S.), Ghrayeb, J.), Chang, E. centre.

Tee, (Chang, N, T,), Gear mouth, R, Sea, (Gallo, R, C,), Wong-stall, F, (Wong-stall, P,), Ko Complete Nucleotide Sea Fence on The Aids Virus, H TLV III (Complete Nucleotide 5eque nce of the AIDS virus.

HTLV-111), Nature, 313.277-284 (1985 )15, Sanchez-Pescador, etc. Science, 227, 484-492 (1985) 16 , Smith, G, L, (Sm1th, G, L,), 77 Keto, M.

(Mackett, M,), Moss, B, (Moss, B,), Natu re, 302゜17, Smith, G, L, (Smith, G, L,), Murphy, Bee.

Earl, (Murphy, B, R,), Moss, B, (Moss, B,) .

(Proc, Natl, Acad, Set, USA, 80.715 5-7159 (1983) 18, Venkatesan, Nis, (Venkate san, S,), baroudy, B, M, (Baroudy, B, M,) +Moss, Bee, (Moss.

B.), Cell, 125, 805-813 (1985) 19, U. Vain-Poplin, Varnish, (Vain-Hobson, S.), Sonigo, Pi -, (Sonigo, P,), Danos, Ou, (Danos, 0.).

Cole, Varnish, (Cole, S.), Allicin, M- (Alizon.

M,), Nucleotide Sea Fence on the AIDS Virus, L.A.V. Nucleotide 5equence of the AIDS vir us, LAY), Ce1l, 40.9-17 (1985) 20, Wire , J., B. (Veir, J.P.), Moss, B.

(Moss, B.), Journal on Vialogy (J.

Virol, ), 48, 530-537 (1983) 21, Schiller, M. Zoller, M.J., Smith, M. ), oligonucleotide-directed mutagenesis on DFF I Fragments Cloned Into M13 Vectors (Oligo Nucleotide-directed mutagenesis of D NA fragments cloned 1nto M2S vectors) Methods in Enzymolog y), 100.488-P S P 5 ueue ueue ■＊　－－－－－－－－１－－←－←■）－1138－－－ト１１１－１１６ 3 -Inside--Akira-mk PCT/FR87100116

Claims (1)

[Claims] 1 at least ・Part of the virus genome, ・Encodes one of the envelope glycoproteins (gp) of the AIDS-causing virus A virus containing the genes and elements that lead to the expression of the glycoprotein in cells. vector. 2 Claims in which part of the virus genome is part of the box virus genome Viral vectors in Section 1. 3. The virus vector according to claim 2, wherein the box virus is vaccinia. . 4. The gene encoding one of the envelope gps encodes gp160 The viral vector according to any one of claims 1 to 3, which is a gene. 5. A claim in which the gene encoding one of the gp is a gene encoding gp41. Viral vectors in ranges 1 to 3. 6 Claim that the gene encoding one of the gp is a gene encoding gp120 Viral vectors in the range 1 to 3. 7 The signal sequence S of the env gene is the g of the envelope of the AIDS-causing virus. The viral vector of claims 1 to 6, which precedes the gene encoding p. . 8 There is an env gene in the gene encoding gp of the envelope of the AIDS-causing virus. The virus according to claims 1 to 7, which is followed by a transmembrane tm sequence of the gene. Svector. 9 Contains a signal sequence and/or transmembrane sequence derived from a heterologous virus Viral vectors according to claims 1 to 8. 10 The signal sequence and/or transmembrane sequence derived from a foreign virus is disrupted. The viral vector according to claim 9, which is derived from a canine disease virus. 11 The gene encoding one of the gp is from less than 5': ・The sign of the env gene null array, ・gp120, ・gP40, ・The entire env gene including the transmembrane (tm) region of the env gene Viral vectors according to certain claims 1-3 and 7-10. 12 The gene encoding the entire env gene is the env gene of the LAV virus. The viral vector according to claim 11. 13. The tm region has been mutated and the Arg codon has been replaced with a He codon. Viral vectors according to claims 7 to 12. 14 Env gene undergoes mutation and protease between gp120 and gp40 Viral vectors according to claims 1 to 13, wherein the cleavage site is removed. -. 15 The DNA sequence encoding the gp molecule(s) is present in the box virus genome. Viral vectors according to claims 1 to 14, which are dependent on gene promoters. . 16 Claim 15, wherein the promoter is a vaccinia gene promoter viral vectors. 17 The DNA sequence encoding gp molecule(s) is 7.5 of vaccinia. The viral vector according to claim 16, which is under the control of the promoter of the K protein gene. ctor. 18 The sequence encoding the gp molecule(s) is present in the vaccinia TK gene. The viral vector according to any one of claims 15 to 17, which is cloned into -. 19 The env gene has undergone at least two mutations, and gp120/gp4 Claim 1 in which the protease cleavage site in the 0 junction region is excluded. The viral vector according to any of items 18 to 18. 20 The sequences encoding the extracytoplasmic region and the intracytoplasmic region contain a C-terminal hydrophobic peptide. 20. The viral vector of claim 19, wherein the viral vector is synchronously fused after deletion of the code. 21 The gene encoding gp40 encodes the hydrophobic N-terminal portion of gp. 19. The viral vector according to any one of claims 1 to 18, which lacks the sequence. 22 The extracytoplasmic and intracytoplasmic regions of gp160 are The wire according to any one of claims 1 to 19, which is synchronously fused after removal. Rus vector. 23 Any of the above claims containing a sequence encoding one of the following proteins: Reka's virus vector. ▲Contains mathematical formulas, chemical formulas, tables, etc.▼ ・S is a signal peptide, ・gp120 is glycoprotein 120, ・J is between gp120 and gp40, Conceptually shows the part where the protease cleavage site has been deleted, ・gp40 is glycoprotein 40, -tm is a transmembrane peptide. 24. Claim 23, wherein the glycoprotein 40 lacks its hydrophobic N-terminus. virus vector. 25 Claims 19 to 24 contain a sequence encoding a protein with the following structure: Any of the following viral vectors: -S-gp40-tm or -S-gp120- In the tm formula, gp40 is a glycoprotein 40 with its hydrophobic N-terminus deleted, S, tm and gp120 have the meanings defined in claim 24. 26 VV. T.G. eLAV1125(9-1), VV. T.G. eLAV112 8, VV. T.G. eLAV1130, VV. T.G. eLAV1131, VV. T.G. eLAV1132, VV. T.G. eLAV1133, and VV. T.G. eLAV1134. Viral vectors selected from. 27 VV. T.G. eLAV1135, VV. T.G. eLAV1139, VV. T.G. eLAV1136, VV. T.G. eLAV1137, and VV. T.G. eLAV1138. Viral vectors selected from. 28 VV. T.G. eLAV1162 and VV. T.G. eLAV1163. Viral vectors selected from. 29. Recombinant DNA corresponding to the viral vectors of claims 1 to 28. 30 Infected with the viral vectors of claims 1 to 28 or claims 1 to 28 A culture of mammalian cells containing the DNA of Section 29. 31. Cultivating the cells according to claim 30 and collecting the produced glycoproteins. A method for preparing the envelope glycoprotein of the AIDS-causing virus, including. 32 Enzyme of AIDS-causing virus obtained by carrying out the method of claim 31 Velope glycoprotein. 33. The glycoprotein of claim 32, further comprising a part of rabies glycoprotein. 34 Viral vectors according to any of claims 1 to 28 and/or claims A vaccine comprising the glycoprotein according to any of Items 32 and 33. 35 Two or more types of viruses with different structures and/or two or more types of glycoproteins with different structures 35. The vaccine of claim 34 containing white matter. 36 Virus VV. T.G. eLAV1136 and VV. T.G. eLAV113 8 or a combination of glycoproteins corresponding to these. Chin. 37 Virus according to any one of claims 1 to 28 or claim 32 or The glycoprotein described in Section 33 is inoculated into living organisms, and the antibodies produced are collected after a predetermined period of time. Antibodies produced against the envelope glycoprotein of the virus that causes HIV.