JPH01179687A - Hiv fused protein - Google Patents

Abstract

PURPOSE:To obtain a gag-env fused protein having both gag antigen and env antigen, by fusing a gag antigen gene fraction containing a range to code p24 of HIV with an env antigen gene fraction containing a range to code gp41. CONSTITUTION:A gag antigen gene fraction containing a range to code p24 of HIV is fused with an env antigen gene fraction containing a range to code gp41 to make the same reading frame. Then development by yeast is carried out to give a gag-env fused protein having both antigenicities of gag-p24 and env-gp41. The prepared gag-env fused protein of HIV has antigenicities of characteristic AIDS virus and env protein and is suitable as a reagent for measuring AIDS-related antibodies and useful for vaccine.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention provides a fusion protein of gag and env antigens of the AIDS virus, which is the causative virus of acquired immunodeficiency syndrome (AIDS), that is, an AIDS virus expressed using genetic modification technology. gag-env fusion protein.

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 is a disease.

Characteristic clinical symptoms exhibited by AIDS patients include carinii pneumonia and Kaboji's sarcoma, which are thought to be caused by rapid decline in human immune function due to AIDS virus infection. AIDS, which exhibits these 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 virus in Europe and Japan. It is feared as an infectious disease.

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. on the other hand,
There are great hopes for a vaccine that can prevent infection, but unlike normal viruses, there are many problems in vaccine development due to severe antigenic variation on the virus surface, and currently there are no effective vaccines. No vaccine has been developed.

In such a situation, the most important countermeasure is to check for blood and infected people, which are the sources of AIDS virus infection, and to prevent new infections from occurring from these sources. In other words, clinical test reagents that can detect whether the AIDS virus is present in samples will play a major role, and the development of such clinical test reagents is being actively researched along with the development of vaccines and treatments. There is. Testing reagents that can directly detect the AIDS virus or its components are not yet commercially available, but antibodies against the AIDS virus can be detected and indirect virus contamination (infection) can be detected.
Several types of clinical test reagents that can detect this have already been developed and are widely available on the market. Today, these test reagents are indispensable in institutions that handle blood, etc.
It is used effectively.

The virus that causes AIDS infection is the LAV [5science, Vol.
, 220p868-871 (+983) and JP-A No. 60-67859, HTL by Gallo et al.
V-m [5science Vol, 224p50f1
503 (+984), and Special Publication No. 61-500987
These viruses and other AIDS-related viruses [e.g. ARV: Levy et al., 5science Vol, 225 p840 (+
084) HIV (human immu)
Nodeficiency virus). In this specification, these various AIDS-related viruses will be collectively referred to as HIV.

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. env consists of gp41 and gp120 glycoproteins, and gp41
gp120 penetrates the membrane, and gp120 protrudes outside the membrane. Inside the virus, there is a core antigen called gag, and three proteins called p14, p24, and p+7. Additionally, there are reverse transcriptases, endonucleases, and proteases, which are encoded within the pol gene.

As for the structure of the gene genome, there are DNA sequences called long terminal repeats (LTRs) at both ends of the provirus.
It has a structure in which genes encoding gag, pot, and env exist between them, and the length of the genome is approximately 0.7K.
bp gene. The entire base sequence of this HIV genome has already been cloned [Shav et al., 5ci
ence Vol, 226 pH651171 (198
4)].

Several examples of attempts to express AIDS virus-related polypeptides using genetic recombination have been reported so far.

For example, N, T., and Chang et al. cut HTV DNA into DNA fragments of about 500 bp (base pairs), inserted this into an expression vector for E. coli, and used
It was expressed as a fusion protein with galactosidase. As a result, we obtained transformed E. coli that expresses a protein that reacts with AIDS and human serum.
228.93-96 (1986)]. However, the AIDS-related antigen substance expressed here is a fusion protein with β-galactosidase, and when used as a test reagent for measuring AIDS-related antigens, it is preferable that it is a pure AIDS-related antigen.

R. A. Kramer et al.
The gene fragment encoding the and pot gene was developed for yeast.
p24.H vector, which is a gag protein. p
+? and p14 protein [5cien
ce 231.1580-1584 (1986).

Ph1lip J, Ban et al. attempted to express the env antigens gp120 and gp41 of the AIDS virus in yeast using GAP (glutaraldehyde-3-phosphate dehydrogenase) and ADH (alcohol dehydrogenase) promoters. reported that the expressed gp41 was not efficiently expressed because it was toxic to yeast within the yeast cells.

On the other hand, SOD (human superoxide desmutase)
In experiments in which they were expressed as fusion proteins with Vaccine 5. 90-101 (1987
)Ko. However, in this case, gp41 is expressed as a fusion protein with SOD, which has no relation to the AIDS virus, so pure expression of gp41 is desired when considering its use as a vaccine or test reagent.

Currently commercially available reagents for measuring AIDS-related antibodies are made using antigens obtained by disrupting cultured viruses, so the production of these reagents requires direct handling of the infectious HIV virus. There are concerns about the risk of virus infection. Even if such an accident occurs to those involved in the work, it is preferable to convert the work into a safe work that does not require handling infectious viruses. That is, it is desired to develop safe and effective measurement reagents or vaccines using non-infectious antigens prepared by techniques such as genetic recombination.

In general, AIDS patients and those infected with the AIDS virus are
Since there are cases where there are antibodies against both the v antigen and the gagJ* antigen, or only one of them, the antibody measurement reagent must be able to detect both of these antibodies.

On the other hand, the main vaccine against AIDS has been e-
The target has been placed on the nv antigen. Recently, it has become clear that among env antigens, gρ41, which is a conserve region, also has a peptide region that is important as a vaccine. For example, Kennedy et al.
The region 735-752 in the v antigen constituent amino acid sequence (gp
We prepared a synthetic peptide containing a peptide (region near the C-terminus of 41) and immunized rabbits with it, which successfully induced effective antibodies that react with the env antigen [
5science Vol, 23+ 1556-1559
(1986)]. In addition, D, D, No et al. prepared 22 different peptides considered to be important regions from the amino acid sequence of the env antigen and immunized them with these, and found that they were able to induce neutralizing antibodies in some of them. is reported. Among them, regions 816-632 and 728-751 (all numbers correspond to the order from the N-terminus of the entire enV antigen amino acid sequence) in the amino acid sequence encoded by gp41 have the ability to induce neutralizing antibodies. is reported. [Journal of Virolor
y Vol, 61No, 6 p2024-2028
(1987)].

Furthermore, it has been reported that in AIDS patients, as their symptoms worsen, antibodies against p24 among antibodies against 83 and antigens decrease [JonathanN,
Weber et al.; Lancet, January
17. pH9-121 (1987)], gag
The presence of p24 antibodies may suppress the progression of AIDS symptoms.

Judging from these circumstances, it is thought that it is more effective for an AIDS vaccine to be made from both env and gag antigens, and similarly, clinical test reagents that specifically detect antibodies against these antigens are considered to be more effective. Developments such as these are also considered to be extremely important.

However, as mentioned earlier, although the current gene recombination technology has succeeded in expressing the 8ag antigen p24, the env antigen gp41 cannot be expressed in pure form, that is, in other forms not related to the AIDS virus. Efficient expression of env antigen in a pure form other than a fusion protein with a peptide has not been successful, and the development of a method for efficiently producing env antigen by genetic recombination is eagerly awaited. Furthermore, in today's world, where both env and gag antigens are considered essential components of vaccines, there remains a problem in terms of the amount of work involved in producing and purifying each of them separately from transformants. It is hoped that genetic recombination technology and culture technology that can efficiently prepare both of these antigens will be developed.

Under these circumstances, the present inventors discovered that HIV
gag antigen gene fragment containing the region encoding 24 and g
When we fused the env antigen gene fragment containing the region encoding p41 into a single reading frame and attempted to express it in yeast, we found that gag-p24 and env
We succeeded in obtaining a gag-env fusion protein that has both antigenic properties of v-gp41. Furthermore, it has been extremely difficult to express the env antigen in a pure form in a recombinant body, but the gag-env fusion protein according to the present invention
We have completed the present invention by discovering that the expressed product env antigen is not toxic to host cells and can efficiently produce the target product like gag antigen. Furthermore, according to the present invention, the HIV
gag with antigenicity of both gag antigen and env antigen
-env fusion protein can be obtained at once, and great effects can be obtained in industrial applications. Further, such env-gag fusion protein is expected to be used as a multivalent subunit vaccine in vaccine development.

That is, the present invention provides a novel gag-env fusion protein of the AIDS virus that is useful for AIDS diagnostic reagents and vaccines. For more details, see the AIDS virus g, which was efficiently expressed using genetic recombination technology.
The present invention provides a fusion protein of ag protein and env protein, and a method for producing the same.

・H]- The HIV gene used in the present invention can be obtained by conventional gene cloning methods from AIDS viruses that have been isolated so far, such as [NATURE Vol. 312. p
166-169 (1984)].

For example) 19/) Virus-infected cell line designated by ITLV-m B (ATCC No. CRL8543)
Mo1t3/HTLV-mB (ATCCNo, CRL
8602) is an example of a virus that can be handled manually. DNA was extracted from these virus-infected cells and the previously reported base sequence of HIV-DNA [Nat
ure Vol, 313 p277-284 (198
5) DNA probes synthesized with reference to et al. and commercially available HTV-DNA probes (Biotech Re
s, Lab.

, Md, , LISA) can also be used to clone HIV-DNA.

The desired en at the position in the AIDS virus gene genome.
Regarding the presence of v gene and gag gene,
Report by Gallo et al. (NATLIRE Vol, 3+3 p2
77-284 (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 Bal-31). Furthermore, if necessary, it is necessary to attach an appropriate linker to the end of the gene of interest to facilitate subsequent genetic manipulation.

In this way, each env gene and g
Isolate the ag gene.

Or HrV-DN that has already been reported as above
Referring to the gene sequence of A, only a gene encoding a polypeptide of a desired region of the required antigen can be synthesized using a DNA synthesizer and used for the preparation of expression plasmids and transformants described below.

Next, the two genes separated and prepared in this manner are fused to form the same reading frame as described below. It is known that gp41 and gp120 exist as envvc antigens (surface antigens) of the AIDS virus, but the genes encoding the fusion peptide of the present invention include
HI that includes at least the entire region encoding gp41
The env gene fragment derived from V is used. On the other hand, gag
Although ρ24, p+7, and p+4 are known as antigens, in the present invention, a gag gene fragment derived from f(IV) containing at least the entire region encoding p24 is used. is, g
By linking the gene encoding ag in the 5J orientation, the target product can be expressed effectively. In addition, since there is no translation initiation codon in the gene encoding gag-p24, if part of the 5' region of the gag p17 gene is removed, the synthetic NA
It is necessary to previously attach a translation initiation codon (ATG) to the 5' end using a suitable linker having a linker containing ATG or ATG so that the translation initiation codon (ATG) is in frame with the 5' end. Alternatively, it is also possible to use an improved plasmid having a translation initiation codon (ATG) downstream of the expression promoter for the foreign gene in the expression vector. An env gene fragment containing a region encoding the env antigen gp41 is ligated to the 3' end of the gag gene so that they are in frame. The stop codon that stops translation is the original stop codon of gp41.

The gene encoding the desired fusion protein thus obtained can be expressed in eukaryotic cells such as yeast and animal cells as hosts. If yeast, among eukaryotic cells, is used as a host, the resulting transformant can be handled very easily, and the desired fusion protein can be prepared relatively easily. Furthermore, when used as a vaccine, from the viewpoint of safety, it is preferable to use yeastshu, which poses almost no danger to humans, rather than using animal cells containing carcinogenic factors as hosts.

The expression vector necessary for expression in such a host needs to be constructed depending on the function of the host cell used.

For example, when using yeast as a host, it is desirable to use a shuttle vector that can be cloned in both E. coli and yeast. The structure of such an E. coli/yeast shuttle vector is that it has a plasmid replication initiation region derived from an E. coli plasmid and drug resistance genes (ampicillin resistance gene, tetracycline gene, etc.) as genes on the E. coli side, and as genes on the yeast side.
It has a yeast-derived replication initiation region (2 μori, ars 1, etc.) and a yeast selection marker gene (leu2, etc.). Furthermore, in order to express the target gene in yeast, it is necessary to incorporate a yeast-derived promoter into the above-mentioned shuttle vector, and such promoters include yeast acid phosphatase (PH05) promoter and glutaraldehyde dehydrogenase-3 promoter. -Lin m
(GAP-DH) promoter and the like are most preferred. By inserting the HrV3aro-env fusion gene downstream of the promoter gene for foreign gene expression in such a shuttle vector, the HI
A plasmid for expressing V gag-env fusion protein is obtained. This plasmid is introduced into yeast to obtain transformed yeast expressing the 11IV fusion protein.

Furthermore, when using cultured cells such as animal-derived cells as hosts, the following methods can be considered. For example, by replacing a portion of a viral gene such as SV40 with the HIV fusion gene of the present invention and introducing it together with a helper virus into a host cell (such as a monkey-derived cell), the fusion protein of the present invention can be expressed. A recombinant virus is obtained. Alternatively, by combining a portion of a viral gene (such as papillomavirus) that can be propagated in a plasmid state within a host cell with an HIV fusion gene and introducing it into the host cell, it is possible to obtain cells that express the target gene in a plasmid state. can. Alternatively, an Escherichia coli/cultured cell shuttle vector can also be used, similar to the shuttle vector when yeast is used as the host cell.

The composition of such an E. coli/cultured cell shuttle vector is a plasmid having a promoter capable of functioning in cultured cells in addition to the E. coli-derived gene incorporated into the yeast shuttle vector. By integrating an HIV fusion gene downstream of the promoter of this shuttle vector, a plasmid for cultured cells capable of expressing the HIV fusion protein of the present invention can be obtained, and when this is introduced into cultured cells, silk The f IV fusion gene can be expressed in cultured cells. As such a plasmid, a commercially available one such as pSVL (Pharmacia Co., Ltd.) can be used.

The desired gag-env fusion protein can be efficiently obtained by culturing the L(IV gag-env fusion protein-expressing transformant obtained as described above) under culture conditions in which the used promoter can function efficiently. can.

The HIV gag-env fusion protein thus obtained is a combination of the original gag protein of the AIDS virus and envy.
It is a novel protein that has white matter antigenicity and is suitable for use as a reagent for measuring AIDS-related antibodies and as a vaccine. That is, the 3ag-env fusion protein of the present invention is suitable for gag-p, which is considered to be particularly important as an antigen for vaccines.
Since it contains all the peptide regions of 24 and env-gp41, it is considered to be very effective in inducing antibodies against HIV. In addition, especially when used as an antigen for use in reagents for measuring AIDS-related antibodies, it can be an excellent antibody measuring reagent that replaces current commercially available products. That is, by using the fusion protein of the present invention, it is possible to specifically and simultaneously detect only antibodies against gag protein and env protein, which are considered necessary for AIDS screening. Also,
With conventional genetic recombination technology, it was not possible to express the env protein gp41 in a pure form, so there was a problem in producing effective antibody test reagents and vaccines through genetic recombination. This problem can also be solved by using fusion proteins.

In addition to the technical progress in terms of quality as described above, the present invention also has a significant economic effect on manufacturers and consumers of such clinical test reagents. In other words, when obtaining two different proteins using conventional genetic recombination technology, each protein must be expressed and expressed separately.
Since it was necessary to culture and purify it, it was a two-fold process, and problems remained in terms of time and cost. However, according to the present invention, both antigens of interest, namely gag
It becomes possible to prepare the protein and env protein in a short period of time and at low cost by culturing and purifying the protein and env protein once. Nowadays, when the problem of AIDS is becoming more serious, the social effects of the present invention are extremely large, and its significance is also significant.

Hereinafter, the present invention will be explained in more detail with reference to Examples in which yeast is used as the expression host, but the present invention is not limited to the following Examples.

Example 1 (1) Construction of plasmid Recombinant plasmid λ81110 in which HIV DNA was cloned [Nature 313 p24 (+
985), U.S. National Insurance 9! Institute of Health (Nationa)
l In5titude of Health:NtH
) obtained from Dr. Gallo] 20 μg was digested with restriction enzymes Sac I and 88III, and approximately 1.4 Kb of the ρ gene encoding the gag protein ρ24 (see Figure 1) was separated by agarose electrophoresis. In addition, the aforementioned plasmid λB++102oH was digested with restriction enzymes BglII and XhoI.
The 1.2Kbp gene containing the gp41 gene of the env protein was cut with agarose electrophoresis in the same manner as shown in Figure 1! Ib) was separated. On the other hand, plasmid ρFB+2 (
(manufactured by Pharmacia) using restriction enzymes Xho I and Sac.
The DNA fragment of 3.2 Kbp was separated by agarose electrophoresis.

Each of the above three gene fragments obtained in this way +0
0ng was added to T ligase reaction solution (66mM Tris-11
cIpH7,6,6,6a+M MgCl2. l0mM
DD T, ImM ATP) using 2 units of T4 DNA ligase at 4° C. for 4 hours. Escherichia coli χ1776 was transformed with this reaction solution according to the method described in Yasutaka Takagi's "Gene Manipulation Experimental Methods" page 161, and colonies grown on an agar medium containing 50 μ3/ml of ampicillin were extracted from "Metabolism" No. 17. Volume, No. 4 “Lipase J 1
Plasmids were prepared according to the method described in 181-89 (1980). As a result, the Xh of pFBI2
Blasmi FGE1 was selected, in which a fusion gene of a gene containing a region encoding HIV gB p24 and a gene containing a region encoding env gp41 was inserted between o I and 5ac I (Fig. 2). reference).

The thus obtained pF GE 1 5μ8 was cut with restriction enzymes Pvu II and
．． The IKbr+DNA fragment was isolated.

About 2.1 kbp gag-e obtained in this way
The entire base sequence of the nv fusion gene fragment corresponds to the entire base sequence of the gene shown in FIG. 3, excluding the 7 base pairs at the 5' end (ATGGATC).

On the other hand, yeast inhibitory acid phosphatase (PI(05)
) Synthetic protein N A (CCAT
GGATCCATGG; synthesized using a DNA synthesizer)
and Xho to the downstream Pvu U site.
[See Figure 4 for inserting the linker]. The thus obtained plasmid pAMB (Xhol) was digested with the restriction enzyme Ball1I and added to a T4 polymerase reaction solution [67mM Tris-HCI.

pH 8, 0, 6, 7mM MgCl2. lomM2
- mercaptoethanol, 330 μM each dATP,
dCTP, dGTP, dTTP, 6.7μ ammonium sulfate; (NHa)3sOz, 6.7μM
The sticky end of the +tXhoI linker was converted to a blunt end by reacting with EDTAI at 37°C for 1 hour. Further, this reaction solution was cleaved with the restriction enzyme Xho I, and a 10 Kbp DNA fragment was obtained by agarose electrophoresis.

These two DNA fragments mentioned above are subjected to a ligation reaction using T4 DNA ligase. As a result, H
A plasmid pGE1 (see FIG. 5) into which the IV gag-env fusion gene was inserted was obtained. The entire base sequence of the structural gene of the gag-env fusion protein expressed in the plasmid thus obtained is shown in FIG. E. coli into which this gag-env fusion protein expression plasmid pGE1 has been introduced is
P-9776).

(2) Expression of fusion peptide p24-p41 by yeast Saccharomyces cerevisiae Al122 as host yeast
(a 1eu2 his4 canl(Cir”))
(Feikoken Joyori No. 312), and this was mixed with YPD medium (2χ polypeptone, I! yeast extract, 2z g/L).
Course) After inoculating the cells into a room and cultivating them overnight at 30°C, the cells were collected by centrifugation. The bacterial cells were washed with sterile water, suspended in 5 ml of a solution of 1.2 M sorbitol and 100 μg/ml thymolyase 60,000 (Seikagaku Corporation), and kept at 30°C for 30 minutes to form spheroplasts. After washing the feroplasts three times with 1.2M sorbitol solution, 1.2M sorbitol, 10mM
CaCl2 and IOmMTris-HCl (pH 7
, 5). Add the plasmid pGE1 prepared in (1) above to this, mix well, and add 0.1
Add M CaCl2 to 10mM 1:aC
I2 and left at room temperature for 5 to 10 minutes. Next to this,
20% polyethylene glycol 4000, 10mM C
aCl2 and 10mM Tris-HCI (pH 7,
5) The solution was added and left at room temperature for 20 minutes. Add 0.21 portions of this mixture to a regeneration medium (22% sorbitol, 2% glucose, 0.7% yeast nitrogen-based amino acids, 2% YPD, 20 μ8/ml) kept at 45°C.
Histidine, 3% agar), mix gently, and add to the previously prepared 1, 2M sorbitol-containing minimal medium (0,7% yeast nitrogen-based amino acids, 2% glucose, 20 μ8/1 histidine, 2% agar) plate. After solidifying the mixture, it was cultured at 30°C to obtain a colony of leucine non-requiring yeast. 20 colonies
Bulkholder minimal medium containing μg/ml histidine (
Toyo et al., J. Bachterof, 113. p
727-738) 10+nl, cultured at 37°C.

After 24 hours, the bacterial cells in the logarithmic growth phase were isolated and placed in a phosphate-free minimal medium (H2PO4 contained in bulk holder minimal medium was replaced with C1,
Additionally, 20μgem I histidine was added)
After suspending 10ml of bacterial cells to approximately 4xlO6cells/ml and continuing culturing at 30°C for approximately 24 hours,
Bacterial cells were collected by centrifugation at 0 rpm for 10 minutes. 1.2M sorbitol, 50mM phosphate buffer? a (p
H7,2), 111 μM 2-mercaptoethanol,
The suspension was suspended in 3 ml of a 100 μg/ml Thymolyase 60,000 solution, gently shaken at 30° C. for 30 minutes to form spheroplasts, and the spheroplasts were collected by centrifugation. This ferroblast was dissolved in 50mM phosphate buffer (pH
7,2) Suspend in 1 ml, add glass beads and stir to destroy bacterial cells. This crushing liquid was rotated at 5000 rpm for 10 minutes.
The mixture was centrifuged for a minute, and the supernatant was examined for antigen production using the Western blotting method described below.

Samples were electrophoresed on a 15% 5DS-polyacrylamide gel and then electrically transferred to a nitrocellulose filter. Soak the filter in 5% skim milk for 1 hour.
) f [monoclonal antibody against V-gagp24 [Japan J, Cancer R
es, Vol, 78. p235-241 (+9
87), kindly provided by Kumamoto University School of Medicine] for 2 hours. The filter was soaked in Tris buffer (50mM Tris,
After washing with 15 M NaCI), it was reacted with an anti-mouse IgG peroxidase-conjugated antibody (manufactured by Bio-Rad) for 1 hour at room temperature. After washing with TBS, color was developed with hydrogen peroxide and diaminobenzidine. As a result, in the yeast extract sample containing pGE1, molecules m6B, 2 and 92.
A broad band of about 80 to 85 daltons was observed between 5 and 85 daltons. This is the gag-env of the present invention.
The molecular weight was almost the same as that estimated from the fusion gene. On the other hand, no such band was observed in the negative control yeast extract sample containing pAMB1 which does not contain the gag-env fusion gene (see Figure 6).

[Brief explanation of the drawing]

FIG. 1 shows restriction enzyme maps of the HT and LV-m genes and the regions encoding gag%pol and env. Figure 2 shows plasmid pFGE1. FIG. 3 shows the entire base sequence of the gag-env fusion protein structural gene encoded downstream of the foreign gene expression promoter in plasmid pGE1. FIG. 4 shows plasmid pAMB 1 (Xhol). Figure 5 shows plasmid pGE1. FIG. 6 is a diagram showing the results of Western blotting of the gag-env fusion protein of the present invention. Ne1201 ATA, GTG, CAG, CAG, CAG, AAC, A
AT, TTG, CTG, AGG, GCT. 11 e-Va l -G l n-G l n-G
l n-Asn-Asn-Leu -Leu-Arg-
A l a-ne 1261 CTC, ACA, GTC, TGG, GGC, ATC, A
AG, CAG, CTC, CAG, GCA. Leu-Thr-VaI-Trp-Gly-1
1e-Lys-Gln-Leu-Gln-A l
a-ne1321 AAG, GAT, CAA, CAG, CTC, CTG, G
GG, ATT, TGG, GGT, TGC. Lys-Asp-Gln-Gln-Leu-L
eu -G Iy-11e-Trp-G Iy-Cy
s-* 1381 GTG, CCT, TGG, AAT, GCT, AGT, T
GG, AGT, AAT, AAA, TCT. Val-Pro-Trp-Asn-Ala-5
er-Trp-Ser-Asn-Lys-5e r-ne1441 TGG, ATG, GAG, TGG, GAC, AGA, G
AA, ATT, AAC, AAT, TAC. Trp-Met-G lu-Trp-Asp-Arg-
G l u -11e-Asn-Asn-Tyr-*
1501 GAA, TCG, CAA, AAC, CAG, CAA, G
AA, AAG, AAT, GAA, CAA. G 1u-5er-G I n-Asn-G I n-
G In-G Iu-Lys-Asn-Glu-G l
n-1260neATT, GAG, GCG, CAA, CAG, CAT, C
TG, TTG, CAA-I-1e-Glu-Ala-G
l n-Gln-H1s-Leu-Leu-Gin13
20ne AGA, ATC, CTG, GCT, GTG, GAA, A
GA, TAC, CTA・Arg-11e-Leu-Al
a-Val-Glu-Arg-Tyr-Leu1380
*TCT, GGA, AAA, CTC, ATT, TGC, A
CC, ACT, GCT-5er-G Iy-Lys-L
eu-11e-Cys-Thr-Thr-Ala144
0*, CTG, GAA, CAG, ATT, TGG, AAT,
AAC, ATG, ACC・Leu-Glu-Gln-1
1e-Trp-Asn-Asn-Met-Thr150
0 * ACA, AGC, TTA, ATA, CAC, TCC, T
TA, ATT, GAA=Thr-5er-Leu-11
e-His-Ser-Leu-l 1e-Glu156
0*, GAA, TTA, TTG, GAA, TTA, GAT,
AAA, TGG, GCA-Glu-Leu-Leu-G
Iu-L, eu-Asp-Lys-Trp-Ala Figure 5 gag gene M6 diagram pAMBl pGE1

Claims (3)

[Claims] (1) Among the gag genes of HIV, at least p24
HIV gag obtained by expressing in eukaryotic cells a fusion gene consisting of a gene fragment containing the entire region encoding gp41 and an env gene fragment containing at least the entire region encoding gp41 of the env gene.
-env fusion protein. (2) The fusion gene is a fusion gene of an approximately 0.9 kbp gene from the PvuII site to the BglII site of the HTLV-IIIgag gene and an approximately 1.2 kbp gene from the BglII site to the Xho I site of the HTLV-IIIenv gene. The fusion protein according to item (1) above. (3) The fusion protein according to item (1) or item (2) above, wherein the eukaryotic cell is the yeast Saccharomyces cerevisiae.