JPH05301895A - Hybrid antigen protein, recombined virus for expressing the same, and its production - Google Patents

Abstract

PURPOSE:To provide the hybrid antigen protein having a virus-constituting protein and the small peptide epitope of a pathogenic virus and useful as a vaccine or a diagnostic medicine. CONSTITUTION:The hybrid antigen protein having the virus-constituting protein and the small peptide epitope of the pathogenic virus is produced by preparing a recombined virus having the gene of the virus-constituting protein and the gene of the small peptide epitope of the pathogenic virus and subsequently allowing the recombined virus to express in the cultured cell.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a hybrid antigen protein having a viral structural protein and a peptide epitope of a pathogenic virus whose constituent amino acids are 300 or less, a recombinant virus expressing the same, and a hybrid antigen protein using the same. It relates to a manufacturing method.

[0002]

2. Description of the Related Art In recent years, peptide epitopes that are important for defense against infection have been found in antigen proteins of various pathogenic viruses, bacteria, protozoa, etc., and their use as vaccines has been attempted (R. Arnon).
and M.D. Shapira, Modern Appr
oaches to Vaccines, Molecu
lar and Chemical Basis of of
Virus and Immunogenecity
(RM Chenock et. Al. Eds.)
p. 109, Cold Spring Harbor
(1984)). However, since these relatively short-chain peptide vaccines generally have low antigenicity, they are used in combination with an adjuvant after being combined with keyhole limpet hemocyanin, and side effects such as inflammation due to the adjuvant. There was a drawback that occurred.

On the other hand, it has been reported that a live vaccine using a recombinant virus is highly safe and exhibits excellent antigenicity (Japanese Patent Laid-Open No. 64-74982, etc.). Therefore,
It is considered that if the peptide epitope corresponding to the synthetic peptide vaccine can be efficiently expressed by the recombinant virus, the drawback of the peptide vaccine having low antigenicity can be improved.

However, according to the experiments by the present inventors, in order to express a small peptide epitope using a recombinant virus, a conventional method for expressing a large antigen protein (Japanese Patent Laid-Open No. 64-74982, etc.) was used. Even if it was used as it was, there was a problem in the stability of the small peptide in cells, and it was not possible to obtain a sufficient expression level.

[0005]

Therefore, the present inventors have found that
Under such conventional technology, as a result of intensive studies to express a peptide epitope of a relatively short chain, a recombinant virus incorporating a gene encoding a viral structural protein and a gene encoding a peptide epitope of a pathogenic virus was developed. It was found that when used, a hybrid antigen protein having a viral structural protein and a small epitope can be efficiently obtained, and the present invention has been completed.

[0006]

Thus, according to the present invention, as a first invention, a viral structural protein (hereinafter referred to as structural protein) and constituent amino acids are 300 or less, preferably 10 to 150 constituent amino acids, A hybrid antigen protein (hereinafter, referred to as hybrid protein) having a peptide epitope (hereinafter, referred to as small epitope) of a pathogenic virus, which is more preferably 15 to 100, and further preferably 20 to 50, is provided.

As a second invention, a gene encoding a viral structural protein expressing the hybrid protein (hereinafter referred to as a structural protein gene) and a gene encoding a peptide epitope of a pathogenic virus having 300 or less constituent amino acids ( Hereinafter, a small-sized epitope gene) is incorporated into a region not essential for the growth of the virus to provide a recombinant virus. Furthermore, as a third invention, a method for producing a hybrid protein using this recombinant virus is provided.

In the present invention, a virus used for producing a recombinant virus (hereinafter referred to as a host virus) may be a virus used in a general gene recombination technique, and examples thereof include vaccinia virus, baculovirus, and avi. Examples include pox virus. It is preferable to use a virus that infects the same species as the animal that is infected by the pathogenic virus from which the peptide epitope was derived. For example, if a peptide epitope derived from human immunodeficiency virus (hereinafter referred to as HIV) is incorporated,
Vaccinia virus that infects humans may be used.

Examples of vaccinia virus include WR strain (J. Virol., 49, 857, (1984)),
Lister strain, temperature-sensitive Lister strain (JP-A-62-44)
178), New York Board of H
Vaccinia vaccine strains such as health strain and LC16m8 strain are exemplified, and examples of baculovirus include Autographa californica (Autographa cal
ifornica), Trichoprussia ni (Trich)
oplusia ni), Rakiburushia Ou (Rac
hiplusia ou), Galeria melonella (Ga
llelliamelloella), Bombix
Bombyx mori and the like, and examples of avipoxvirus include ATCC VR-25.
1, ATCC VR-250, ATCC VR-22
9, ATCC VR-249, ATCC VR-22
8, Nishigahara strain, NP strain (chicken embryo pigeon pox poison Nakano strain) and the like.

The structural protein gene used in the present invention is not particularly limited as long as it functions as a viral structural protein. For example, vaccinia virus major outer membrane antigen p37 (J. Virol., 39,
903 (1981)), vaccinia virus hemagglutinin (Virology, 150, 451 (198).
6)), vaccinia virus Ag35 antigen (J. Vir)
ol. , 181, 671 (1991)) and the like. Moreover, these protein genes may be modified (including amino acid deletion, increase, and change) as long as they express a protein functioning as a structural protein.

Of such protein genes, from the viewpoint of immunogenicity, viral outer membrane proteins are preferable, and viral outer membrane proteins that are expressed on the surface of infected cells are more preferable. Specific examples of such a gene include vaccinia virus major outer membrane protein p37 (J. Virol., 58, 757 (198).
6)), vaccinia virus hemagglutinin and the like. It is also preferable to use a structural protein of a virus belonging to the same genus as the virus to be incorporated.

The size of the structural protein gene to be incorporated is not particularly limited as long as the virus from which the structural protein is derived is a virus of the same species as the virus to be incorporated (hereinafter referred to as the host virus), it does not affect the growth of the host virus. However, when a structural protein gene derived from a virus heterologous to the host virus is used, it must be large enough to allow the host virus to grow, and a gene that is too large cannot be incorporated. In such a case, the size of the gene is usually 10,000 bases or less, preferably 2,000 bases or less.

The gene encoding a small epitope used in the present invention is an epitope of a pathogenic virus and has 300 or less constituent amino acids, preferably 10 to 15 amino acids.
0, more preferably 15 to 100, still more preferably 2
Genes encoding 0 to 50, for example, a gene encoding HGP30 of HIV (Pro
c. Natl. Acad. Sci. USA, 84, 29
51-2955 (1988)) and a gene encoding HIV V3 (Proc. Natl. Acad. Sc).
i. USA, 85, 1932 (1988)) and the like.

In addition, these protein genes are modified (including amino acid deletion, increase, and change) as long as they express those that function as small epitopes.
May be obtained from a natural virus, a part of a cDNA of a viral gene, or a synthetic one.

The hybrid protein of the present invention is produced by preparing a recombinant virus in which the above-mentioned constituent protein gene and a small epitope gene are incorporated into the above-mentioned host virus, and expressing this in an appropriate host cell. Specific examples of such hybrid proteins include hybrid proteins of p37 protein derived from vaccinia virus and HGP30 protein derived from HIV, p37 protein derived from vaccinia virus and H
Examples thereof include hybrid proteins with IV-derived V3 protein.

The general method for producing the recombinant virus and hybrid protein of the present invention will be described below.

(Preparation of First Recombinant Vector) The first recombinant vector contains a constituent protein gene used for preparing a target hybrid protein. When the constitutive protein gene and the small epitope gene are inserted into the host virus by homologous recombination, some of the genes that are not essential for the growth of the host virus must be located at both ends of the structural protein gene. Therefore, when the virus to be incorporated is a virus of a species different from the host virus, it is necessary to make a part of the gene region not essential for the growth of the host virus exist at both ends of the constituent protein gene.

The vector used as a material for preparing the first recombinant vector is not particularly limited, but for example, pBR32
2, plasmids such as pBR325 and pUC18, phages such as λ phage and M13 phage, pHC79 (Gene, 11, 291 and (198
Cosmids such as 0)) are exemplified.

The first recombinant vector can be prepared according to a conventional method (JP-A-62-44178, JP-A-1-285198, JP-A-1-168).
279 publication). For example, when a protein derived from vaccinia virus is used as a constituent protein and vaccinia virus is used as a host virus, J. Virol. Meth
ods, 2, 175-179 (1981), using a vaccinia virus-derived DNA prepared according to the method described above, a part of the vaccinia virus-constituting protein gene and its peripheral portion are cut out with an appropriate restriction enzyme, It can be incorporated into a vector.

(Preparation of Second Recombinant Vector) The second recombinant vector is prepared by inserting or adding the desired small epitope gene into any part of the structural protein gene in the first recombinant vector thus prepared. To make. Upon insertion or addition, both the structural protein gene and the small epitope gene must be connected in the correct reading frame for amino acid translation, and the promoter must be included so that both genes are not destroyed.

When the small epitope gene is added immediately upstream of the start codon of the structural protein gene, the start codon is required in the most upstream of the small epitope gene, and the small epitope gene is provided immediately downstream of the stop codon of the structural protein gene. If added, it is necessary to remove the stop codon of the structural protein gene and create a stop codon at the most downstream of the small epitope gene.

(Preparation of Third Recombinant Vector) A marker gene for facilitating selection of recombinant virus is inserted into the second recombinant vector by a conventional method,
A third recombinant vector is created.

The marker gene is not particularly limited,
For example, the β-galactosidase gene (Molecula
r And Celler Biology, 5, 34
03 (1985)), the Ecogpt gene (J. Vir.
ol. , 62, 1849-1854 (1988)) and the like. The marker gene is used in a state of being linked to the downstream of the promoter.

The promoter linked to the marker gene is not particularly limited as long as it functions in the virus to be incorporated. For example, as a promoter expressed in vaccinia virus, a vaccinia virus encoding a 7.5K polypeptide is used. Gene promoter (hereinafter 7.5K)
A promoter of a vaccinia virus gene encoding a 19K polypeptide (hereinafter, referred to as 1)
9K promoter), the promoter of the vaccinia virus gene encoding 11K polypeptide, and the like, and those expressed in baculovirus include the baculovirus gene promoter encoding baculovirus polyhedrin, the baculovirus 10K poly Examples include baculovirus gene promoters encoding peptides, and those expressed in avipoxvirus include avipoxvirus gene promoters encoding avipoxvirus thymidine kinase, 7.5K promoter, and 19K promoter.
Examples thereof include promoters.

The marker gene linked to the downstream of the promoter is inserted in the vicinity of the vaccinia virus component protein gene to which the second small epitope gene has been inserted or added to prepare the third recombinant vector. In constructing these first, second and third recombinant vectors, an Escherichia coli system that can be easily genetically manipulated may be used.

(Construction of Recombinant Virus and Production of Protein) The third recombinant vector obtained by the above-mentioned method was introduced into cells previously infected with virus to obtain vector DN.
A recombinant virus is constructed by causing homologous recombination between A and the gene of the viral genome. In constructing the recombinant virus, it may be carried out according to a conventional method, for example,
The third recombinant vector is introduced into the virus by the calcium phosphate coprecipitation method, the liposome method, the microinjection method, the electroporation method, etc., and the obtained virus population containing the recombinant virus is cultured on an appropriate medium.

Plaques are formed by a method suitable for the marker gene inserted in the third recombinant vector to obtain a target recombinant vaccinia virus candidate strain. When the β-galactosidase gene is used as the marker gene, after forming plaques, an Eagle MEM containing halogenated indolyl-β-D-galactosidase (hereinafter referred to as bluogal) and agar is overlaid, and Plaques that stain blue after night may be used as a candidate recombinant vaccinia virus candidate strain.

The method for selecting the target recombinant virus from these candidate strains is to purify plaques by using a hybridization method using the target small epitope gene as a probe, or to target the small epitope. An inom assay using an antiserum or a monoclonal antibody against E. coli may be performed. A host cell is infected with the recombinant virus thus obtained and cultured to produce a hybrid protein.

The cells used here are not particularly limited as long as they are infected by the virus serving as the host. When the host virus is vaccinia virus, for example, TK ^- 143 (derived from human osteosarcoma), FL (derived from human amnion), Hela (derived from human cervical cancer), KB (derived from human nasopharyngeal cancer), CV-1 (from monkey kidney), BSC-1
(Derived from monkey kidney), RK13 (derived from rabbit kidney), L929
(Derived from mouse connective tissue), CE (chicken embryo), CEF (chicken embryo fibroblast), and the like. When using baculovirus, for example, Spodoptera frugiperda (S
S from podoptera frugiperda)
F9 cells and the like are exemplified, and when an avipox virus is used, CE (chicken embryo), CEF (chicken embryo fibroblast) and the like are exemplified.

Since the hybrid protein thus produced is usually present in a large amount in the host cell during the life of the host cell, the cells may be collected and crushed to be recovered. The hybrid protein can be purified by appropriately combining well-known conventional methods such as salting out, gel filtration, ion exchange and separation by affinity column chromatography, high performance liquid chromatography, and fractionation by electrophoresis.

[0031]

INDUSTRIAL APPLICABILITY According to the present invention, thus, a small epitope can be expressed as a hybrid antigen protein with a structural protein, this protein can be used as an antigen of a component vaccine, and a recombinant virus can be used as a live vaccine. Expected to be used.

[0032]

EXAMPLES The present invention will be described in more detail with reference to the following examples. (Example 1) Vaccinia virus major outer membrane antigen p37
Preparation of the first recombinant plasmid pHP37 by cloning the gene and its surroundings (see FIG. 1) Preparation of vaccinia virus genomic DNA was first carried out by the vaccinia virus LC16mO strain (clinical and virus, 3 (3) 13).
-19 (1975), hereinafter referred to as vaccinia virus mO strain) is cultured according to a conventional method, and then the J. Virol. Met
Hods, 2, 175-179 (1981).

This genomic DNA was cleaved with the restriction enzymes PstI and HindIII to obtain p37 gene-containing 4
A 452 bp DNA fragment was recovered. pUC18 was cleaved with restriction enzymes PstI and HindIII, and the DNA fragment obtained above was inserted into the first recombinant plasmid p
HP37 was made. Confirmation of pHP37 is Viro
l. 179, 247-266 (1990), and confirmed the restriction enzyme site based on the nucleotide sequence.

(Example 2) Synthesis of HGP-30 gene and production of second recombinant plasmid having p37 gene and HGP-30 gene (1) Plasmid pG containing the latter half of p37 gene
Preparation of H37 (see FIG. 2) The pHP37 obtained in Reference Example 1 was cleaved with the restriction enzymes HpaI and BglII, and the latter half of the p37 gene, about 12
A 60 bp DNA fragment was recovered. This fragment was treated with Klenow to make it blunt-ended, and then pBR322 (Cold Spring Hub) completely digested with a restriction enzyme DraI.
or Symposium, 43, 77, (197
9)) to obtain a recombinant plasmid pGH37. pGH37 has a single DraI site at a position overlapping with the stop codon of the p37 gene.

(2) Plasmid pGH3 by insertion of synthetic DNA corresponding to HGP-30 gene into pGH37
Preparation of 7HGP (see FIG. 2) DNA encoding HGP-30 gene (SEQ ID NO: 1)
Was synthesized using a DNA synthesizer. This synthetic DNA is
It has a HindIII site in the center. The pGH37 gene obtained in (1) above was treated with a restriction enzyme DraI, and the synthetic DNA encoding HGP-30 obtained above was inserted to obtain a recombinant plasmid pGH37HGP. pGH37
HGP was treated with the restriction enzyme SspI to generate p37 gene and H
A DNA fragment near the GP-30 gene was obtained, and the nucleotide sequence of this fragment was determined by the method of Meshing et al. (Nucl. Acid.
s. Res. , 9, 309-321 (1981)), pGH37HGP was a restriction enzyme Dr.
It was found that the stop codon was lost between the p37 gene and the HGP-30 gene located downstream thereof due to the treatment with aI, and a codon encoding tyrosine was obtained.

(3) Construction of recombinant plasmid pKPHGP30 by adding p37 gene rear peripheral sequence and p37 gene first half sequence (see FIGS. 3 and 4) The restriction enzyme Hind of pGH37HGP obtained in (2) above was used.
It was digested with III and the restriction enzyme SspI to recover a 361 bp DNA fragment containing the latter half of the HGP-30 gene. In addition, the pHP37 obtained in (1) above was converted to the restriction enzyme Ps.
It was cleaved with tI and the restriction enzyme SspI, and a DNA fragment of about 1250 bp containing the rear sequence of the p37 gene was recovered. Next, pUC18 was digested with the restriction enzyme HindIII and the restriction enzyme P.
The 361 bp DNA fragment and the approximately 1250 bp DNA fragment which had been treated with stI and recovered were inserted to prepare a recombinant plasmid pHPRear.

Similarly, pGH37HG obtained in (2) above
91 bp D containing the first half of the HGP-30 gene, which is obtained by cleaving P with the restriction enzymes HindIII and SspI.
The NA fragment was recovered. In addition, pHP3 obtained in (1) above
7 was cleaved with restriction enzymes KpnI and SspI to give p
A DNA fragment of about 1050 bp containing the forward sequence of 37 genes was recovered. Next, pUC18 was digested with the restriction enzyme HindI.
The 91 bp DNA fragment and the approximately 1050 bp DNA fragment recovered by treating with II and the restriction enzyme KpnI were inserted to prepare a recombinant plasmid pHKFront.

Recombinant plasmid p thus obtained
HPRear with restriction enzyme PstI and restriction enzyme HindI
II and cleaved to the latter half of the HGP-30 gene and p
Approximately 1510 bp including the sequence of the rear part of 37 genes
Was recovered. In addition, recombinant plasmid p
Restriction enzyme PstI and restriction enzyme Hind for HKFront
After cutting with III, a DNA fragment of about 1140 bp containing the sequences of the first half of the HGP-30 gene and the front of the p37 gene was recovered. The restriction enzyme PstI for pUC18
Was treated with the restriction enzyme KpnI and the two recovered fragments were inserted to obtain a recombinant plasmid pKPHGP30.

(4) Construction of second recombinant plasmid pHPHGP30 by adding p37 gene forward sequence (see FIG. 5) Recombinant plasmid pKPHGP30 obtained in the above (3)
Digested with restriction enzymes PstI and KpnI to give p3
A DNA fragment of about 2650 bp containing a partial sequence at the rear of the 7 gene and the p37 gene was recovered.

Further, the recombinant plasmid pHP37 obtained in the above (1) was treated with the restriction enzyme KpnI and the restriction enzyme HindI.
After cutting with II, a DNA fragment of about 1890 bp containing a partial sequence around the front of the p37 gene was recovered. treating pUC18 with the restriction enzymes PstI and HindIII,
Insert the two recovered fragments into the recombinant plasmid pH
PHGP30 was produced.

Example 3 Third Recombinant Plasmid pHPHGP3 by Incorporating β-Galactosidase Gene into Second Recombinant Plasmid pHPHGP30
Preparation of 0Z (see FIG. 6) (1) pNZ76 in which β-galactosidase gene was integrated downstream of 7.5K promoter of vaccinia virus
Preparation of β-galactosidase gene is described in Gene, 28, 12
7-132 (1984) described plasmid pMA001
Was digested with the restriction enzyme BamHI and recovered. Plasmid p with polylinker downstream of 7.5K promoter
AK8 (Japanese Patent Laid-Open No. 64-74982) is used as BamHI
And the previously recovered β-galactosidase gene was inserted to obtain a recombinant plasmid pNZ76.

(2) Second recombinant plasmid pHPH
Construction of Third Recombinant Plasmid pHPHGP30Z by Incorporation of β-Galactosidase Gene into GP30 pNZ76 obtained in (1) above was restricted with HindIII
Was digested with the restriction enzyme SmaI to recover a DNA fragment in which the 7.5K promoter was linked to the β-galactosidase gene, which was further treated with Klenow to obtain a DNA fragment of about 4 Kbp. The recombinant plasmid pHPHGP30 obtained in Reference Example 2 (4) above was treated with a restriction enzyme SnaBI,
The about 4 Kbp DNA fragment obtained above was inserted to prepare a recombinant plasmid pHPHGP30Z.

Example 4 Preparation of Recombinant Vaccinia Virus RK-13 Cultured in a 25 cm ² Culture Bottle
Vaccinia virus WR strain was added to the cells at 0.1 p. f. u.
45 min after inoculation at a rate of 10 cells / cell, 10 μg of Example 3
2.2 of the recombinant plasmid pHPHGP30Z obtained in
Dissolve in ml of sterilized water and use Hitaka et al. (protein, nucleic acid, enzyme, 2
7, 340- (1985)), a DNA-calcium phosphate coprecipitate was prepared, and 0.5 ml thereof was added dropwise to infected RK-13 cells. 30 minutes, 37 ℃, 7% C
The mixture was allowed to stand in an O ₂ incubator, and 4.5 ml of Eagle MEM containing 5% fetal bovine serum was added. After 3 hours, the culture solution was exchanged, and the cells were cultured for 48 hours at 37 ° C. in a 7% CO ₂ incubator, and each cultured cell was freeze-thawed 3 times to obtain a virus solution containing a recombinant.

For selection of recombinants, RK-13 cells cultured in 10 cm Petri dishes were inoculated with the above virus solution, and 30 minutes later, Eagle MEM containing 0.8% agarose and 5% fetal bovine serum was used. After stacking and culturing for 3 days, Eagle MEM containing 0.8% agarose and 0.5 mg / ml of BruoGal (manufactured by BRL) was stacked on the infected cells, and 1
After 4 hours, the virus was extracted from the blue-colored plaque with a Pasteur pipette, suspended in phosphate-buffered saline containing 2% gelatin (hereinafter referred to as PBS), and part of it was subjected to dot hybridization.
Spotted on nylon or nitrocellulose membrane, the rest was stored at -20 ° C.

The spotted membrane was treated with a 0.5N sodium hydroxide aqueous solution for 10 minutes and a 1M Tris-hydrochloric acid buffer solution for 5 minutes, and then treated with 1.5M sodium chloride, 0.5M.
It was treated with M Tris-HCl buffer for 5 minutes. 2 times SSC (1
Double SSC is 0.15M sodium chloride, 0.015M
It was saturated with sodium citrate) and baked at 80 ° C. for 2 hours.

Thereafter, this was subjected to 4 times SET (0.6 M sodium chloride, 0.08 M Tris-hydrochloride, 4 mM EDT).
A, pH 7.8) -10 times Denhardt-0.1%
The mixture was treated with SDS mixed solution at 68 ° C. for 2 hours. 4x SET-10x Denhardt-0.1% SDS-
_{0.1% Na 4 P 2 O 7} -50μg / ml denatured salmon sperm D
HGP-labeled with ³² P by NA and kaination
30 synthetic genes were added and hybridized at 68 ° C. for 14 hours. After washing, the membrane and X-ray film were overlapped and autoradiography was performed to select spots where the film was blackened.

The virus solution (virus amount moi = 3 × 10 ⁻⁶ ) corresponding to the blackened spot was RK-again.
13 cells were inoculated, and 30 minutes later, 0.8% agarose, 5
% Of fetal bovine serum was added to the MEM, and after culturing for 3 days, 0.8% agarose and 0.5 mg / ml of bleugar eagle MEM were added to the MEM, and after 14 hours, the plaque stained blue was described above. Purification was repeated until all the plaques that appeared were blackened by dot hybridization. The virus thus obtained was the recombinant vaccinia virus of interest and was named v37HGP30.

(Comparative Example 1) Construction of recombinant vaccinia virus having no small epitope HGP-30 gene without p37 gene (1) Recombinant virus having structural protein p37 gene having small epitope HGP-30 gene Construction of plasmid (see FIG. 3) pAK8 was treated with restriction enzymes SalI and EcoRI and synthesized by a DNA synthesizer (SEQ ID NO: 2,
Has SalI site on 5'upstream side and Ec on 3'downstream side
A HGP-30 synthetic gene having an oRI site) was inserted to prepare a recombinant plasmid pAK8HGP30. In pAK8HGP30, the vaccinia virus TK gene is disrupted, and the HGP30 synthetic gene having a start codon and a stop codon before and after is linked to the downstream of the vaccinia virus 7.5K promoter.

(2) Construction of recombinant vaccinia virus Instead of pHPHGP30Z as a recombinant plasmid, the recombinant plasmid pAK8HG obtained in (1) above was used.
A recombinant vaccinia virus was constructed in the same manner as in Example 4 except that P30 was used and the virus was selected with 5-bromo-2′-deoxyuridine, and the resulting recombinant vaccinia virus was named vTKHGP30. did.

Example 5 Expression of Recombinant Vaccinia Virus in Infected Cells RK-13 cells grown in Eagle MEM containing 5% fetal calf serum on a tissue culture chamber slide were treated with m.p.
o. i. = 1 of the recombinant vaccinia virus v of the present invention
37HGP30, or vTKHGP constructed in Comparative Example
Each was inoculated with 30 and left at 37 ° C. for 1 hour, then the virus solution was removed, the cells were washed with Eagle MEM, and Eagle MEM containing 5% fetal bovine serum was added. 1
After 6 hours, remove Eagle MEM and gently wash with PBS,
After air-drying, the cells were fixed by treating with acetone at room temperature for 5 minutes.

Anti-HGP-30 monoclonal antibody (manufactured by Viral Technologies Inc.) was used as the primary antibody, and fluorescein isothiocyanate-conjugated anti-mouse I was used as the secondary antibody.
The indirect fluorescent antibody method using the gG antibody (manufactured by TAGO) was used to observe the specific issue by fluorescence using a fluorescence microscope. As a result, the recombinant vaccinia virus v37HGP30 of the present invention showed a large amount of small epitope HGP-.
Recombinant vaccinia virus vTKHGP3 expressing 30 but lacking the structural protein gene of Comparative Example
It was found that 0 did not express the small epitope HGP-30 at all.

Example 6 Confirmation of Hybrid Protein Expression in Recombinant Vaccinia Virus-Infected Cells Using Western Blotting RK- grown in Eagle MEM containing 5% fetal calf serum in a 10 cm ² culture plate. 13 cells, m.p.
o. i. = 1 of the recombinant vaccinia virus v37HGP30 or vaccinia virus W constructed in Example 4
Rormo strains were inoculated and left at 37 ° C for 1 hour, then the virus solution was removed, and the cells were removed using Eagle ME.
After washing with M, Eagle MEM containing 5% fetal bovine serum was added. After 16 hours, remove Eagle MEM and lightly PBS
The cells were collected by washing the cells in a centrifuge tube, centrifuging at 3000 rpm for 5 minutes.

Next, 100 μl of lysis buffer (N
Aureure, 227, 680 (1970)) was added to resuspend the cells, and the cells were treated at 100 ° C. for 5 minutes. Then, using the supernatant obtained by centrifugation at 15,000 rpm for 10 minutes as a sample, Western blotting was performed according to a conventional method. The primary antibody used is the same as that used in Example 5. As a result of Western blotting, a clear band of about 40 Kd was observed in v37HGP30-infected cells, whereas no band was observed in the vaccinia virus used as the parent strain. This revealed that the hybrid protein in which the HGP-30 protein was added to the p37 protein was expressed in v37HGP30-infected cells.

[Brief description of drawings]

FIG. 1 is an explanatory view showing a method for producing a recombinant plasmid pHP37.

FIG. 2 is an explanatory view showing a method for producing a recombinant plasmid pGH37HGP.

FIG. 3 is an explanatory view showing a method for producing a recombinant plasmid pHPrear and a recombinant plasmid pHKFront.

FIG. 4 is an explanatory view showing a method for producing a recombinant plasmid pKPHGP30.

FIG. 5 is an explanatory view showing a method for producing a recombinant plasmid pHPHGP30.

FIG. 6 is an explanatory view showing a method for producing a recombinant plasmid pHPGHP30Z.

[0054]

[Sequence listing] SEQ ID NO: 1 Sequence length: 94 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence ATAGCGTAC ATCAAAGGAT AGATGTAAAA GACACCAAGG AAGCTTTAGA GAAGATAGAG 60 GAAGAGCAA AACAAAAGTA AGAAAAAGGC TTAA 94

[0055]

[Sequence Listing] SEQ ID NO: 2 Sequence Length: 104 Sequence Type: Nucleic Acid Number of Strands: Double Strand Topology: Linear Sequence Type: Other Nucleic Acid Synthetic DNA Sequence TCGACATGT ATAGCGTACA TCAAAGGATA GATGTAAAAG ACACCAAGGA AGCTTTAGAG 60 AAGATAGAG GAAGAGCAAA ACAAAAAGTA GAAA
AAGGCT TAAG 104

Continuation of front page (51) Int.Cl. ⁵ Identification number Office reference number FI technical display location A61K 39/275 9284-4C C12N 7/01 C12P 21/02 C 8214-4B // C12N 15/39 ZNA 15 / 62 15/86 (C12P 21/02 C12R 1:92)

Claims (3)

[Claims] 1. A hybrid antigen protein comprising a virus structural protein and a pathogenic virus peptide epitope having 300 or less constituent amino acids. 2. A recombinant virus in which a gene encoding a viral structural protein and a gene encoding a peptide epitope of a pathogenic virus whose constituent amino acids are 300 or less are incorporated into a non-essential region for virus growth. 3. The method for producing the protein according to claim 1, which uses the recombinant virus according to claim 2.