JPH0269194A - Mixed antigen particle composed of hbc or hbe antigen and hiv-neutralizing epitope - Google Patents

Abstract

PURPOSE:To obtain an antigen effective against HIV by bonding a gene coding HIV-neutralizing epitope to a structural gene such as HBc and expressing the obtained fused structural gene in a host cell by the aid of an expression vector. CONSTITUTION:An HIV epitope (a) which is a mixed antigen particle is prepared by using a peptide having an amino acid sequence of formula corresponding to the amino acid sequence numbers of from 308 (Asn) to 327 (Ile) of env-gp120 of HIV (AIDS-relating virus). A fused structural gene (c) is produced by bonding a gene coding the component (a) to a structural gene (b) composed of HBc or HBe polypeptide. The component (c) is introduced into an expression vector of E.coli, etc., and an E.coli (HB101), etc., are transformed with the obtained expression vector to form a transformant (d). The transformant (d) is cultured in a medium and the obtained microbial cells are extracted to obtain a mixed antigen particle containing HBc or HBe antigen and an HIV- neutralizing epitope.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to antigen particles comprising an HBc or HBe antigen and an HIV-related peptide, and a method for producing the same. Specifically, the present invention relates to hybrid antigen particles, which are genetically recombinant expression products, consisting of HBc antigen or HBe antigen and an HIV neutralizing epitope (an antigen epitope that induces an antibody having neutralizing activity against HIV), and a method for producing the same.

Acquired immunodeficiency syndrome (AIDS) is a relatively new immune disease that was first reported to infect humans around 1981, and no effective preventive vaccine or treatment has been established to date. It's a bright color. Characteristic clinical symptoms of AIDS and laziness include carinii pneumonia and Kaboji's sarcoma.
These are thought to be caused by the rapid decline in human immunity due to AIDS virus infection. This...
AIDS, which exhibits similar symptoms, is spreading mainly in Africa and America, and once symptoms develop, the fatality rate within two years is said to be over 70%, and today it is a serious and deadly viral infection in Europe and Japan as well. feared.

Research and exploration of methods for treating AIDS are underway at various research institutions in many countries.
To date, no effective treatment method has been found; however,
There are high hopes for a vaccine that can prevent infection, but unlike normal viruses, there are many challenges in developing a vaccine due to the severe antigenic variation on the surface of the virus, and currently there are no effective vaccines available. No vaccine has been developed.

The virus that causes AIDS infection is LAV [5science, Vol. 22, as reported by Montani et al.
0 p868871 (1983), and JP-A-1986-
No. 67859] and l-I T l-V by Gallo et al.
-Ill [, 5science Vol, 224 p.
500-503 (+984), and Special Publication 1986-500
987], but today these and other AIDS-related viruses [e.g. ARVs:
Levy et al., 5science Vol. 1.225 p84
0 (1984)] are collectively referred to as HIV (human
In this specification, AIDS-related viruses with various names are collectively referred to as HTV.

HIV, the AIDS-causing virus, belongs to retroviruses, and its virus particles have a diameter of about 1000 angstroms, and the outside of the particles is covered with a membrane consisting of a lipid bilayer derived from the cell membrane of the host's T4 lymphocytes. A glycoprotein called env is present in the membrane. en
v consists of gp'11 and gp120 glycoproteins, g
p41 penetrates the membrane, and gp+20 protrudes outside the membrane. Inside the virus there is a core antigen called gag, p+, 4, p24 and p17.
There are three proteins called Furthermore, reverse transcriptase, endonuclease, and protease are present, and these are encoded within the po13i gene.

As for the structure of the gene genome, there are DNA sequences called long terminal repeat sequences (LTR) at both ends of the provirus.
It has a structure in which genes encoding gag, pol, and env exist, and the genome length is approximately 9.7 mm.
This is the Kbp gene. The entire base sequence of this HIV genome has already been cloned [Shaw et al., 5
science Vol, 226 pH65-1171 (
1984)].

Research on vaccines against AIDS has mainly focused on the env region, and searches for neutralizing regions using synthetic peptides are underway. As a result of synthesizing a large number of peptides thought to have antigenicity, immunizing animals with them, and examining the virus-neutralizing ability of the antiserum, peptides with neutralizing ability were found at several locations.

[David D, Ho et al., Journal
of Virology, vol.

101, No. 6 p2024-2028. (198
7) TholIas J, Pa1ker et al. Proc.
, Na1. , to cad, sci, vol, 85p193
2-1936 (1988).

child. The futile search like ') is due to the fact that it is extremely difficult to obtain monoclonal antibodies capable of neutralizing the AIDS virus.

Furthermore, this suggests that the immunogenicity of the region that induces neutralizing ability is quite low.

However, at present, vaccines using synthetic peptides have not yet been put into practical use, and one of the reasons for this is low immunogenicity. In order to overcome that problem,
Attempts have been made to incorporate neutralizing epitopes into naturally occurring particulate macromolecular proteins using genetic recombination techniques. Previous reports have shown that HBs antigen and HBc antigen, which are hepatitis B virus-related antigens, can be used as carriers to increase the immunogenicity of neutralizing epitopes. ) The gene encoding the neutralizing antigen of the poliovirus was incorporated into the lBs3ft gene, exposing the neutralizing antigen on the surface of the 5-particle, and the possibility of use as a multivalent vaccine was demonstrated.
evo1. 233 p472-474 (1986
), Journal of Virology
.

vol, 62 No, 5 p1836-1893 (1
988)1. Regarding the HB c antigen, its ability to induce cell-mediated immunity involved in humoral immunity has attracted attention in addition to its particle-forming ability, and its area has also been determined.
R. Milich et al. Journal of
Immunology vol, 139 p12
23-1231 (+987)], and the immunopotency f! is synthesized by combining the peptide of that region with the peptide of the BreS region. - When examined, the anti-pre-S antibody was significantly increased compared to that alone, and the effect of the l-I B c-derived peptide became clear [David R, Mil.
ich et al. Nature vol, 329p547-5
49 (1987>1) Furthermore, a gene encoding a neutralizing epitope of Oral Disease was ligated to the 5' end of the HBcil gene, and the fusion protein was expressed in animal cells. Antiserum against the product was prepared. When examining its neutralizing activity, it was reported that it was 100 times more effective than ordinary synthetic peptides [B, EC1arke et al.
Nature vol, 330 p381-384
(1987)].

Purpose of the Invention Under these circumstances, the present inventors identified four sites on the env protein of a monoclonal antibody that has neutralizing activity against HrV, and combined the gene encoding the epitope with the HBc or HBe gene. As a result, we succeeded in expressing a sperm-like fusion protein containing H1-neutralized shrimp and completed the present invention.

That is, the present invention provides novel antigen particles that have an epitope of the HIV antigen exposed on their surface that can induce antibodies having neutralizing activity against l-11V. The hybrid antigen particles of the present invention obtained in this way are
It is expected to be an effective antigen as a vaccine against HTV.

0 ν (-HIV neutralizing epitopes in this specification include antigenic epitopes capable of inducing antibodies having neutralizing activity against HIV.

The neutralizing monoclonal antibody against I-(TV) used in the present invention is based on the rMedic antibody established by Shuji Matsushita et al.
al IffimunologL 3.14 (+987
)], identifying the epitope of the monoclonal antibody (hereinafter referred to as 05β antibody) is an important part of the present invention. The gtll system has been used as a technique for antibody epitope mapping. This system is R
cDNA made from an antibody by Ichard et al.
It was developed with the promise of cloning A [
Proc, Natl, Acad, Sci, vo
l, 80 pH941198<1983)], λ
A gene to be screened is inserted into the EcoR1 site of the 1acZ gene of gtll, E. coli is infected, and the expression product is detected using an antibody.

The HIV gene used in the present invention can be obtained by conventional gene cloning methods, such as [Nature Vol. 312. p16
6169 (1984) 1 etc.

For example, I'! Virus-infected cell line designated by 9/HTLV-IIIB (ATCC No. CRL8543)
, Malt3/I(TLV-[[B (ATCCN
o, CRL8602) is an example of an available virus. DN from these virus-infected cells.
A was extracted and the previously reported HIV-DNA salt sequence [Nature Vol, 313 p277-
284 (1985) etc.] or a commercially available HIV-DNA probe (Bi
It is possible to clone HIV-DNA using Otech Res, LabMd, USA).

Where in the genome of the AIDS virus is the target e?
Regarding the existence of nvi and gagm genes, there is a report by Gallo et al. [Nature Vol. 13 p. 27
7-284 (1985)], and with reference to this, the gene is cut with a restriction enzyme suitable for the desired gene isolation, and if necessary, an endonuclease (
It can be prepared by deleting the gene with Bad-31). Also, if necessary, use a suitable linker.
It is also necessary to attach it to the end of the desired gene to make it easier to perform subsequent genetic manipulations.

In this way, each env gene of interest is isolated. Or 1-II which has already been reported as above
Referring to the V-DNA (7) 3ft gene sequence, synthesize only the gene encoding the polypeptide of the desired region of the required antigen using a DNA synthesizer and use it to prepare the expression plasmids and transformants described below. I can do it.

The enV′ii gene obtained as described above was transferred to DNAS
e, λgt1 was fragmented with endonuclease or exonuclease, and EcoRI linkers were attached to both ends.
When the expression product was detected using a 0.5β antibody, the region around amino acid position 320 of the env region was determined. Also, er+v territory 1
When Peptide 3 of 4308-327 was synthesized and its reactivity with 0.5β antibody was examined, it specifically reacted. In the present invention, this sequence is used as a preferred example of an HIV neutralizing epitope. In addition, other regions that induce neutralizing antibodies or cell-mediated immunity against HIV have been reported, and these regions can also be used as the HIV neutralizing neutralizing ate-1 in the present invention.

Genes encoding these peptides are connected or inserted into appropriate positions of the gene encoding HBc or IIBe. That is, a gene encoding an HIV neutralizing peptide is connected upstream (5' end side) of the structural gene of HBc or HBe, or a gene encoding an HIV neutralizing epitope is inserted in the middle of the structural gene of HBc or HBe.

Furthermore, it is possible to insert the gene 3 encoding the I (IV neutralizing epitope) into a total of two or more locations on the 5° side of the HB c or HBe structural gene and along the way. When integrating the encoding gene into the structural gene of HBc or HBe, it is necessary to combine the two genes so that the codons for amino acid translation of both genes do not shift. is inserted into an expression vector of Escherichia coli, yeast, or animal cells and expressed.The protein of the present invention thus expressed in the host cell forms particles, and moreover, contains the epitope recognized by the 0.5β antibody. It is thought that the hybrid antigen particles of the present invention are exposed on the particle surface.When an animal is immunized with the hybrid antigen particles of the present invention, a dramatic increase in immunogenicity can be expected compared to a synthetic peptide that has not been made into particles. , in the present invention, HBc or HBe
As shown in FIG. 6, the HBe structural gene is the 3' side 1. of the HBc structural genes. ) This is the gene fragment from which the region indicated by the line has been removed.

The region indicated by the ':IA line in FIG.
When using the hybrid antigen particles of the present invention as a vaccine, HBe f is a gene encoding HBe f which is less likely to incorporate DNA into the expressed particles.
From the viewpoint of safety, it is more preferable to prepare the hybrid antigen particles of the present invention using the iI synthetic gene.

One strategy to enhance the immunogenicity of specific epitopes at
There are ways to increase budding density. The simplest method is to increase the concentration of the immunogen.9 In the hybrid antigen particles of the present invention, millions of unit peptides constitute particles with a diameter of 30 nm in a close-packed structure. The evitope density on the particle surface is considered to be extremely high. This particle structure is the first feature of the present invention.

4. One of the essential requirements for an IDS vaccine is not only the ability to induce humoral antibodies to inactivate free viruses, but also force-mediated immunity to prevent cell-to-cell infection. Regarding cell-mediated immunity, it is known that the nuclear proteins of influenza, influenza, and rabies induce cytotoxic T cells9. Children have this ability, and this is the second feature of the present invention.

The present invention will be explained in more detail by referring to Example 1 below.

Recombinant plasmid λ BIIIO [Nature vol.
314 p277-284 (1985), Rice (III
National Institute of Health
tute of Heal↑h: NIIT) Obtained from Dr. Gallo] 10μqe restriction enzyme 5Sall and It in
The gene fragment of approximately 2.4 kb encoding the env protein gp120p was separated by cleavage with dTH and agarose electrophoresis. On the other hand, pUc19 (manufactured by Takara Shuzo Co., Ltd.) 2μ9 was cleaved with 5alI and old ndll+. These gene fragments are reacted at 16° C. for 30 minutes using a 10100n DNA ligation kit (manufactured by Takara Shuzo Co., Ltd.). Transform Escherichia coli HBIOI using the method described in Chapter 8 of Genetic Manipulation Experimental Methods J, edited by Yasutaka Takagi, and collect colonies grown on agar medium containing 100 μg/ml of ampicillin, Metabolism, Vol. 17, No. 4. No. 81-89
(1980). As a result, Sa encoding gp120
l It11ndI[[pUC- with lti fragment inserted
gpI20 was obtained.

This pUC-gp120 50μ9 was digested with 5all restriction enzymes.
Cut with old ndlll and env by agarose electrophoresis.
An approximately 2.4 kb gene fragment encoding the protein gp120 was isolated.

This gene fragment was transformed into 201M Tris-) (CI pH
7,5,1,5mMnC12, bovine serum albumin (1
DNase I (DNA
lng/+sl per 10 μg) at 24°C 10-30
It was allowed to react for a minute. The specimens were treated with phenol and precipitated with ethanol. This gene fragment was added to a 20d TJDN^polymerase reaction solution [0.1 unit T4 DNA polymerase,
200 gM-dATPdcTP・dGTP・dTTP
, 67mM Trig-)1cI (pH 8,6), 6.
7mM MgCl2.10mM 2-mercaptoethanol, 16.711M (N11.)2SOa)
After reaction at 37°C for 130 minutes, phenol treatment and ethanol precipitation were performed.

This DNA was ligated using a DNA ligation kit (Takara Shuzo Co., Ltd.
and 10'C for 130 minutes. Did you precipitate the reaction solution with ethanol? &, Digested with restriction enzyme EcoRI.

The reaction solution was run on a 38% polyacrylamide gel, and genes corresponding to 100-300 bp were extracted. This gene fragment was ligated to the EcoRI arm of λgt11 vector DNA (Stratagene) using a DNA ligation kit, and 1n vitro packaging was performed using a Stratagene kit.

E. coli (Y1090) cultured from the phage solution overnight
was infected and grown for 3 hours at 42°C on agar plates containing ampicillin 50μ9 with soft agar, and 10mM
A nitrocellulose filter soaked in IPTG and air-dried is placed on soft agar to induce expression for 37"C4 hours.

Wash the filter with cleaning solution (7ffiM Tris-■C
1pH7,2,151)mM NaCl>, 5%
Incubate with skim milk solution at room temperature for 2 hours. Add 0.5β antibody to the solution [Shuzo Matsushita et al., Medical I
mmunology, volj p14 (1987
)] to give Igq/ml and react at 4°C overnight. After that, wash 3 times with washing solution, and wash with reaction buffer (phosphate buffer pH 7, 2, 0,005X Tween
Biotinylated anti-mouse IgG in 201° fetal bovine serum)
Antibody (manufactured by Tabo) was added at 1μ9. /1 at room temperature for 2 hours. After that, wash 3 times with washing solution.
Peroxidase-conjugated avitin (2μ9/III) adsorbed at 7°C for 2 hours is reacted at 4°C for 1 hour. Then, wash 3 times with washing solution and color development solution [3 cod/11 (RP color development reagent (manufactured by Bio-Rad) dissolved in methanol and add 5 times the volume of washing solution (containing 0.6 J/ml hydrogen peroxide solution)] 3 React. Phage 2 clone was extracted from the plaque corresponding to the position where the blue color developed. Experiment 2 "Preparation of λ phage", Yasutaka Takagi (ed.), "I1 Yunko Manipulation Experiment Method"
(page 11) and Experiment 3 “Extraction of λ phage DNA” (1
The phage DNA was exported, cut with the restriction enzyme EcoRI, and run on an 8% polyacrylamide gel. A gene fragment of approximately 150 kj p and approximately 290 bp was extracted.

Hc of the old 3wp19 (manufactured by Takara Shuzo Co., Ltd.)
It was inserted into the oRI site in both directions, and Ippondo DNA was prepared according to the description in "Proteins, Nucleic Acids, and Enzymes" Vol. 29, No. 4, 294-306 Shin (1984), and The base sequence was determined using ``3 Sequencing Kisoto''. As a result, these genes
v polypeptide amino acid numbers 301-347.295
It turned out to be a gene fragment encoding -387.

Therefore, using the restriction enzyme Mva I located at position 320, it was investigated whether the 0.5β epitope (the epitope to which the 05β antibody is specific) exists before or after it.
pUC-gp120 was cut with the restriction enzyme Alu I, run on an acrylamide gel, extracted into a gene fragment of about 690 bp, and inserted into the Sma I site of pUc18. The plasmid was treated with restriction enzymes^1uI, MvaI, Dde
I (see Figure 1), and gene fragments of approximately 70 bp and approximately +50 bp were extracted from polyacrylamide gel.

This gene fragment was added to a 20Δ 74 DNA polymerase reaction solution [0.1 unit T4 DNA polymerase, 200 j'
M d, ATP, jcTP, dGdingPdTT1', 6
7mM Tris-11CI (pif8.6), 6.
7thM MgCl2, 10n14 2-mercaptoethanol, 16.7mM (NH4) 2304 Trowel 3
7"c, 30 minutes W11 reaction, phenol treatment, and ethanol precipitation. This DNA was reacted with 1μ9 EcoR1 linker for 130 minutes at 10°C using a DNA ligation kit (manufactured by Takara Shuzo Co., Ltd.).The reaction solution was The ethanol-precipitated fragments were digested with the restriction enzyme EcoRI.The reaction solution was run on an 8% polyacrylamide gel to extract each gene.This gene fragment was
Eco of t11 vector DNA (Stratagene)
The RI arm was linked with a DNA ligation kit, and in vitro using a Stratagene kit.
RO packaging was done 3 times.

The phage solution 3-Large M...(Y]090) cultured overnight
was infected and grown on agar plates containing ampicillin 50μ9 with soft agar for 3 hours at 42°C.
A 2L cellulose filter coated with MIPTG and air-dried is placed on soft agar and induced expression is performed at 37°C for 54 hours.

The filter washing solution (7+nM Tris-tlc
l pH 7, 2゜150 IIM NaCl), wash with 5
% skim milk solution at room temperature for 2 hours. O5β antibody was added to the solution at a concentration of 1βg/'1, and 4
Incubate overnight at °C. After that, wash 3 times with washing solution, and use reaction buffer (phosphate gi solution p I+ 7, 2.0.005%
T+reen20, 1% tallow serum) is reacted with biotinylated anti-mouse IgG antibody (manufactured by Tabo) at 1119/1111 at room temperature for 2 hours. Then wash 3 times with cleaning solution.
Extract 1/100 amount of E. coli in reaction buffer in advance? 1! (
Peroxidase-conjugated avitin (2 μ9/ml) adsorbed with Bioland (Bioland) for 2 hours at 37°C is reacted at 4°C for 1 hour. After that, wash 3 times with washing solution and use coloring solution [3mg
/ III HRP coloring reagent (manufactured by Bioland)
Dissolve it in methanol and add 5 times the amount of washing solution (0.64/m
1 (containing hydrogen peroxide solution)) to react. However, positive Zulag could not be detected from these gene libraries. Therefore, it was found that if the gene was cut at amino acid number 320, 0.5β would no longer react.

Furthermore, in order to narrow down the Ebit-7 of 0.5β, env
Amino acid number 308-327 (NNTRKSIRI [
crawlRGPGRAFVTI;Asn-^sn-Thr-A
rg-Lys-3er-11e-Arg-11e-Gl
n-ArgGly-Pro-Gly-Arg-^1a-
20 amino acids of Phe-Val-Thr-11e) were synthesized using a peptide synthesizer (430A> manufactured by Applied Biosystems).The synthesized bebutide was fixed in a 96-well immunoplate overnight at 4°C, and washed with a washing solution (phosphate buffer ,0005% Twel,!n20)
05β (I μg/ml) was allowed to react at room temperature for 2 hours. Thereafter, the plate was washed with a washing solution and reacted with an alkaline phosphotase-conjugated anti-mouse TgG antibody at room temperature for 1 hour, and after washing, color was developed using a 2-osphosphotase-based tablet. As a result, 0
5β specifically reacted with this peptide.

Plasmid pHBv (see Japanese Patent Application No. 57-145093) containing the entire DNA sequence of HBV was digested with restriction enzyme Rsal.
The approximately 6.3 kbp HB c 3ft gene fragment including the pre-0 region was extracted from agarose gel. An Xho I linker was ligated to the gene fragment to create pAcYc1
77 into the Xho I site to obtain pAHBc.
pAI (Bc) was partially cleaved with the restriction enzyme Xho I at the Xho I site located at the N-terminus 1111 of HBc, the ends were made blunt with Frenow fragment, and Ec
The oRI linker was added to the plasmid pAI+Rc0.
The resulting pAIIBcQl was cut with the restriction enzyme EcoRI and digested with Ba131, and then a 5alI linker was ligated to obtain pAIiBc. When we examined the nucleotide sequence of the deleted portion, we found that 110 head regions were deleted, as shown in Figure 2. pAHBc was cut with restriction enzyme Xho LSal I, and the HBc gene was extracted from agarose gel. The gene fragment was made blunt-ended using the Frenow fragment and H
Expression vector p for E. coli is created by ligating a coRI linker.
Oct25 [Osamu Chisaka et al., Gene vol, 60 p.
183-189 (1987)] into the EcoRI site to obtain pOcTllBc.

On the other hand, pUCX, in which the Sma1 site of ptlc18 was converted to Xho I, was cut with the restriction enzyme XhoL SaiD to insert the previous HB c gene 3, and pUcllBc was obtained f4.
In order to bind the 05β epitope to the N-terminal side of HBc, Applied Biosystems' D
NA synthesizer 381. DNA was synthesized using Its complementary 1) annealing NA and pUcHBc to 5
Opened with alI, old nd ■ (inserted into J.) This inserted DNA has a 5III & 1 site, so the presence of that site confirms whether an epitope has been inserted; The Xho 1 site of the plasmid was
This was converted to coRI to obtain pUclI]VIIBc in which a 0.5β epitope was inserted into the N-terminus of )lBc. Cut the plasmid with restriction enzyme F and coRI and HI
Extract the V-HBc gene and create expression vector 11 for E. coli.
Insert into Ecol 1 site of OCT and pOcTII
1nlBc was obtained.

In order to insert a 0.5β epitope at position 40 of the HBc peptide, the DNA shown in FIG. 5 was synthesized using a DNA synthesizer 381 manufactured by Applied Biosystems. pOcTHBc was cut with restriction enzyme Stu I,
This annealed DNA was inserted into pOcTHBc(I
IIV) was obtained. ConopOcTt(Bc (old ■) and p
OctTIIIVIIBc with restriction enzyme P3t1.5tuI
About 4.1 kbp derived from pOcTllBc ()IIV) and pOc'rHIVl (derived from Bc (7
) Approximately 1.4 kbp was extracted from the 77'/0-sgel and the gene fragments were combined to create pOcTHIVI+
Bc (old ■) was obtained.

Site-directed mutagenesis was performed to convert the nucleotide sequence encoding the 179th arginine of 1 (Bc) into a stop codon.
The gene fragment encoding the HB c iff gene was extracted with agarose gel and inserted into the 5alI site of M13IIp18amb. 413ambllBc was obtained. The plasmid was converted into large intestine IT3 (TGI) and the present DNA was prepared as J1. In order to mutate the base sequence of HBc, NA was synthesized as shown in Figure 3. H if mutated
It was designed to create a Kpn I site in the Bc gene.

This D along with synthetic NA to avoid amber mutation
Annealing NA to single-stranded M]3Ilp18ambllBc and elongating the complementary strand with polymerase, transforming E. coli (1 (B2154)), and confirming the Kpn I site to obtain M13ambHB, which has undergone the desired mutation.
I got e. This M13ambmHBe is converted into restriction enzyme Bgl.
A 0.4 kbp gene fragment encoding HB e was extracted from coarse gel. On the other hand,
pOcTHBc and pOcTHIVHBc with restriction enzyme r
The gene fragments were cut with 3gI and each gene fragment of about 46 kbp was extracted. By combining these genes, pOcTHB
e and pOcT[IIVt(Be) were obtained.

pOcTI(IVlllc, pOcTt[Bc (I(
IV), pOcTII +Vl(Be (old ■), pO
cTIIIVII]lle, pOcTIIBc and pOc
THBe (see Figure 6) was transformed into Escherichia coli (HBIOI), and the colonies were cultured overnight. 1 of the culture solution
00 times the volume of the medium, and after culturing for 2 hours, add 3-β inhibitory anhydride (IAA) to a final concentration of 40μ.
g/ml and cultured for 5 hours. The culture medium was collected, and 1/10 volume of the culture medium was crushed with a solution (501M phosphoric acid #Il).
Add street solution, 0.1% Triton 100> and glass beads, and stir for 10 minutes. The extract was centrifuged and the supernatant was purified using Guinabot's 1 (Be RIAKIT's Hse
The beads on which the VC body is immobilized are reacted at room temperature overnight.
Cleaning solution (phosphoric acid yI street solution, 0.005% Tween20
) and reacted with 0.5β (Iμq/++I) at room temperature for 2 hours. Thereafter, the plate was washed with a washing solution and reacted with an alkaline phosphotase-conjugated anti-mouse IgG antibody at room temperature for 1 hour. After washing, the plate was colored using a phosphotase substrate tablet. As a result, 0.5β specifically reacted with the extract of E. coli transformed with a plasmid containing the HIV gene (Table 1).

500uQ of the extract from 60% to 20% of 11.5 liters
density gradient centrifugation (30,000 rpm, 16 hr)
), fractionated with 12I-, and examined the reactivity of these 05β with IBeRTA. A peak was detected at 40% b. From these results, it can be seen that a lattice reacting with 05β is formed.

Table 1 (Note) pOc'rHBc (IIIV), which represents the structural gene incorporated into this 19 expression vector, was added using the restriction enzyme EcoR.
The gene encoding HBc(l]TV) was extracted with DNA polymerase, blunt-ended with DNA polymerase, and Sal I linkers (manufactured by Takara 15 Zo Co., Ltd.) were ligated to both ends. Yeast expression vector pYG100 E Kizai et al., Jo
Urnal of Virology, 61.
No, 11 p3543-3549] with restriction enzyme 5al
r, dephosphorylated with alkaline phosphatase, and ligated the previous gene to pYGIIBc (tllV).
(Fig. 7), Saccharomyces cerevisiae AlI22 [a 1eu2 his4 Ca
nt (Cir+)] (Feikoken Joyori No. 312) and Satucharomyces cerevisiae Al122pho80
(gX engineering iJt Kume 7'r No. 509) was used to inoculate 100 ml of YPD medium <2
After culturing overnight in C, the cells were centrifuged and washed with 211 ml of bacterial collection water, followed by 1.2 M sorbitol and 100 g.
9/ml Zymolyase 60,000 (manufactured by Higaku Kogyo)
The cells were suspended in 51 ml of a solution of 100 ml and kept at 30°C for about 30 minutes to form spheroplasts. The spheroplasts were then washed three times with a 1.2 M sorbitol solution.
2M sorbitol, LOmM CaCl2 and 10
Solution of mM Tris-l+cl (pH 7, 5) 60
The suspension was suspended at 0Δ and aliquots of 0Δ were dispensed into small test tubes. This was combined with the previously prepared recombinant plasmid pYGHBc.
(HIV) and pYGloo were added at 10 μ9 each.
Mix thoroughly and add 0. Add 3Δ of IM CaCl2 residue to a final concentration of 10 mM CaCl2 and bring to room temperature for 5-1
It was left for 0 minutes. This was then supplemented with 20% polyethylene glycol 4D l) 0, 10mM CaCl2 and 1
0++M Tris-11cI (pit 7.5) solution was added in 11 portions, mixed, and brought to room temperature for about 24 hours.

Add 0.2mM each of this mixture to regeneration medium (22z sorbitol, 2x glucose, 1), 7χ yeast nitrogen base amino acids, 2JYr'D, 20
μ9/1 histidine, 3x agar), mix gently, and add previously prepared minimal medium containing 1 and 2M sorbitol (0.7x yeast nitrogen-based amino acids, 2x glucose, 2eu97ml histidine, 2x
Seven sets were layered on agar (agar) plates, solidified, and cultured at 30°C to obtain colonies of non-leucine auxotrophic yeast. This colony was transferred to a bulk boulder containing 20 μ9/1 histidine.
mini? Mumedium [Higashie et al., J. Bacteral
, 113p727-738] to obtain the transformed yeast Satucharomyces cerevisiae.
Spread 1 liter of histidine on a bulk holder minimum medium agar plate and culture at 30°C to form colonies. Isolate the bacterial cells from the colonies, inoculate them into bulk holder minimum medium 1O+al containing 20μ9/ml histidine, and incubate at 30°C.
Cultivate at. Approximately 24 hours later, the bacterial cells in the logarithmic growth phase were centrifuged and electrophotographed, and then transferred to phosphate-free minimal medium (
KlhP contained in bulk holder minimum medium
Otf! -W exchanged with XCI and further 20μg/Ill
Bacteria #, number approximately 4 x 10 in N0ml of water to which histidine was added
The cells were suspended at 6 cells/+nl and cultured at 30° C. for about 24 hours, followed by centrifugation at 4000 rpm for 10 minutes to collect the cells.

These bacterial cells were treated with 1.2M sorbitol, 50I phosphoric acid (pH 17,2), 14mM 2-mercaptoethanol, 100 μq/ml Zymolyase 60.
000 solution 31 and gently shaken at 30°C for 30 minutes to form spheroplasts, which were collected by centrifugation.

The spheroplasts of each of these IX)-Lydon X-10
03 added 50mM phosphate buffer (pH 7,2) 1
The cells were suspended in mM and glass beads were added and stirred to destroy the bacterial cells. These extracts were centrifuged at 15,000 rpm for 10 minutes, and the supernatant was assayed by EIA using a 1ine RIA KIT and 0.5β Sanderch method.

It reacted specifically (Table 2).

Table 2 Sex pOcTt(IVI(Bc and pflcTllBc (1
11V) was transformed into Escherichia coli (HBl, 01), and the colonies were cultured overnight. The cultured medium was added to 100 times the volume of the medium, and after culturing for 2 hours, 3-β intranoquatic acid assoside (iAA) was added to a final concentration of 40μ9. /1 and cultured for 5 hours. Collect bacteria from 50ml of the culture medium and crush 1ml of 7 hard (50mM cherry phosphate solution, 0.1%)
Add Lydon 100) and glass beads and let stand for 10 minutes. Mix 300Δ of the extract with an equal amount of complete adjuvant or incomplete adjuvant.
Three rabbits each were immunized with 00A four times at one week intervals. Three weeks later, the animals were immunized again, and blood was collected one week later. The serum was conjugated with the 0,5β evit-1 peptide synthesized in Example (1), presumably a peptide at the same position of HIV 2 env-gp120 (As ++-Lys-Thr -V
a l -Leu-Ar1<-1l e-Me 1.
-1. e u-14e tSer-Gly-11is-
V; Low Phe-11is-3er-(Iis-Tyr
When -Arg-Pro) was reacted with Ko1-shitapre), it reacted specifically with the 0.5β Evidose peptide (HIVI) of both blood d+, but did not react with the )IIV2 peptide. . Note that the serum before immunization did not react with any of the peptides.

[Brief explanation of the drawing]

FIG. 1 is a diagram showing the genome structure, restriction enzyme map, and 0,5β antibody epitope of HTLV-III-cDNA. Figure 2 shows the base sequence of the 5' end of the HBc gene and H
The amino acid sequence of the N-terminus of Bc is shown. Figure 3 shows a DNA fragment synthesized to convert the 149th arginine codon of HBc into a stop codon. ? Figure A4 shows the DNA fragment synthesized to attach the 0.5β epitope to the N-terminus of )I B c. Figure 5 shows a DNA fragment synthesized to insert the 05β epitope into the 5tuT site of HBc. Figure 6 shows various structural genes incorporated into the expression plasmid for E. coli (p(H:T) and the names of the expression plasmids incorporating these structural genes. Figure 7 shows the expression plasmid for yeast pYGIIBc (H
The structure of IV) is shown. html

Claims (11)

[Claims] (1) A hybrid antigen particle which is a genetically recombinant expression product and consists of a fusion polypeptide of an HBc or HBe polypeptide and an HIV neutralizing epitope. (2) The HIV neutralizing epitope is HIV env-g
The hybrid antigen particle according to item (1) above, which has a partial amino acid sequence of p120. (3) The HIV neutralizing epitope is HIV env-g
The hybrid antigen particle according to item (2) above, which is a peptide comprising the amino acid sequence of Gly-Pro-Gly-Arg from p120 amino acid sequence number 319 to 322. (4) The HIV neutralizing epitope is HIV env-g.
Amino acid sequence number of p120 is 308 (Asn) to 3
27 (Ile), which is the following amino acid sequence.
) The hybrid antigen particle described in section 2. [There is a gene sequence] (5) HBc or HBe characterized in that a fusion structural gene in which a gene encoding an HIV neutralizing epitope is linked to the HBc or HBe structural gene is expressed in a host cell using an expression vector, and then recovered. A method for producing hybrid antigen particles consisting of a fusion peptide of an antigen and an HIV neutralizing epitope. (6) The method according to item (5) above, which is a fusion structural gene in which a gene encoding an HIV neutralizing epitope is linked upstream (5' end) of the HBc or HBe structural gene. (7) HIV in the middle of HBc or HBe structural gene
The production method according to item (5) above, which is a fusion gene into which a gene encoding a neutralizing epitope has been integrated. (8) The method according to item (5) above, which is a fusion structural gene in which a gene encoding an HIV neutralizing epitope is linked upstream (5' end side) of the HBc or HBe structural gene and in the middle of the structural gene. (9) The gene encoding the HIV neutralizing epitope is
IV(7) The production method according to item (5) above, which is a part of the gene encoding env-gp120. (10) The gene encoding the HIV neutralizing epitope is
The method according to item (9) above, which is a gene fragment containing a gene encoding the amino acid sequence of Gly-Pro-Gly-Arg from 319 to 322 in env-gp120 of HIV. (11) The gene encoding the HIV neutralizing epitope is
Said No. 10 (10) is a gene encoding the following peptide.
) The manufacturing method described in section 2. [There is a gene sequence]