JPH06205672A - Production of chimeral protein having antigen site of surface antigen protein of japanese encephalitis virus and hepatitis b virus and recombinant baculovirus therefor - Google Patents

Abstract

PURPOSE:To provide a method for mass production of chimeral protein for polyvalent vaccine for virus, and to provide recombinant baculovirus therefor. CONSTITUTION:Firstly, recombinant baculovirus is produced by incorporating the nonessential domain of the genome of baculovirus with a DNA obtained by fusion between part of the cDNA coding the surface antigen protein of Japanese encephalitis virus and the DNA coding the surface antigen protein of hepatitis B virus. This recombinant baculovirus is then cultured to manifest a polyvalent vaccine. Thus, the objective chimeral protein usable as the polyvalent vaccine can be mass-produced.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a novel chimeric protein produced by recombinant DNA technology. More specifically, it relates to a chimeric protein having antigenic sites of surface antigen proteins of two different viruses, Japanese encephalitis and hepatitis B.

[0002]

2. Description of the Related Art As a vaccine against Japanese encephalitis, a vaccine containing inactivated Japanese encephalitis virus as an active ingredient has been used so far, and healthy mouse brains were vaccinated with the Japanese encephalitis virus Nakayama Research Institute strain and developed mice. The brain was aseptically collected from the above, purified and inactivated by the alcohol-protamine method to obtain an inactivated Japanese encephalitis virus vaccine stock solution (National Institute of Health Sciences, Alumni Association, "Japanese Vaccine", 2nd edition, Showa era). Published by Maruzen Co., Ltd. on January 20, 1977). In such a vaccine production method, a large amount of Japanese encephalitis virus itself is handled, which is extremely dangerous for the person in charge of vaccine production and the production cost is also high.

On the other hand, hepatitis B vaccine is a surface antigen protein (S protein or HBs) purified from the blood of a patient.
The particles are also referred to as Ag). Since this manufacturing method also requires a large amount of patient blood, there is a high risk of virus infection for the person in charge of vaccine production, and the risk of contamination with live virus cannot be abandoned. Research is progressing to develop safe and inexpensive vaccines using recombinant DNA technology.
Succeeded in producing a vaccine comprising BsAg in yeast (Proc. Natl. Aca. Sci. USA.
80 , 1-5 (1983), A.S. Miyanohar
a). Electron micrographs showed that this yeast-made vaccine had the same 22 nm diameter particles as the blood-derived vaccine. Further, HBsAg could be expressed in cultured cells using vaccinia virus or insect virus vector by recombinant virus technology (Nature, 298 , 347-350 (198).
2), G.I. L. Smith et al. Virol. 63 ,
1549-1557 (1989), R.S. Lanford
Etc.).

On the other hand, also in the Japanese encephalitis vaccine, it was possible to express the surface antigen protein of the Japanese encephalitis virus (sometimes referred to as JE-E) in cultured cells by using vaccinia virus or insect virus vector by recombinant virus technology. (Japanese Patent Laid-Open No. 64-74982, Japanese Laid-Open Patent Publication No.
-285198). In addition, recombinant DNA technology has made it possible to develop a vaccine that protects against multiple infectious diseases with a single immunization. Construction of recombinant vaccinia virus with multiple antigens (Gene, 68 , 1-10)
(1988), B.I. E. H. Coupar et al.) And expression of chimeric proteins with multiple antigenic sites (BIOTEC
HNOLOGY, 3 , 323-326 (1985),
P. Valenzuela et al.) Have been tried. However, when actually producing a chimeric protein, there were many cases where the protein itself could not be obtained due to the higher-order structure of the protein having no antigenicity or due to the degradation by the proteolytic enzyme of the host.

Then, the inventors have succeeded in expressing a chimeric protein having an antigenic site of the surface antigen protein of both Japanese encephalitis virus and hepatitis B virus by recombinant vaccinia virus (Human Science Promotion Foundation). Published 1990 Public-Private Joint Project Research Report). However, when the recombinant vaccinia virus was used, the expression level of the chimeric protein was low,
We could not expect a sufficient effect as a vaccine.

[0006]

Therefore, the present inventors have found that
Under such conventional technology, as a result of intensive studies aimed at developing a bivalent vaccine for Japanese encephalitis virus and hepatitis B virus, a part of DNA encoding the surface antigen protein of Japanese encephalitis virus was found to be on the surface of hepatitis B virus. When a gene fused with a DNA encoding an antigen protein is expressed by a recombinant baculovirus, a large amount of a chimeric protein having the antigenicity of both Japanese encephalitis virus and hepatitis B virus is produced, and the chimeric protein is further cultured. They found that they were secreted neatly, and completed the present invention.

[0007]

Thus, according to the present invention, there are provided a recombinant baculovirus expressing a chimeric protein having antigenic sites of surface antigen proteins of both Japanese encephalitis virus and hepatitis B virus, and a method for producing the same. Provided. In the present invention, the virus used for the production of recombinant baculovirus is not particularly limited, and for example, Autographa californica (Auto) can be used.
grapha californica, Trichoplusia ni, Rachibrushia ou, Galleria melonellell
a), or Bombyx m
ori) and the like. Among these viruses, Autographa californica (hereinafter abbreviated as AcNPV) is preferable. These viruses have been extensively studied in the past, and samples can be obtained from many sources, including the Arvovillas Research Unit at Yale University in Connecticut, USA.

The cDNA encoding the surface antigen protein of Japanese encephalitis virus is, for example, the Nakayama Yoken strain or JaO.
It can be prepared using an Ar strain (Arvoviras Research Unit, Yale University, Connecticut, USA) and a Sagayama strain (the same place). For example, the above Sagaya
cDNA5037 prepared from ma strain is disclosed in JP-A-64-
Although disclosed in the example of No. 74982, in the present invention, as long as it has substantially the same function as the above-mentioned cDNA, it is a modified cDNA (that is, one having a nucleotide sequence substituted, inserted or deleted). It may be. of course,
The amino acid sequences may be modified to different extents as long as they have substantially the same function. A cDNA encoding a surface antigen protein of hepatitis B virus has been cloned in Escherichia coli, for example, pBRHBad.
r72 (Nucleic Acid Research,
11 , 4601-4610 (1983)).

In producing a recombinant virus, first, a first recombinant vector incorporating a DNA region that is non-essential for the growth of baculovirus is produced. In this case, it is necessary to allow a promoter that functions in the baculovirus to be present in the region, and it is preferable to insert a synthetic linker having an appropriate restriction enzyme cleavage sequence downstream of the promoter. The term "non-essential DNA region for growth" as used herein refers to a region that does not substantially affect the growth of the virus even if it receives a mutation due to the insertion of foreign DNA.

The promoter functioning in the baculovirus may be any promoter having a nucleotide sequence which can effectively function as a promoter in a transcription system possessed by the virus regardless of whether it is synthetic or natural. Examples of the promoter include a promoter of a gene encoding polyhedrin of baculovirus.

The first recombinant vector can be prepared by a conventional method (for example, JP-A-60-37988, JP-A-61-2587) and, for example, as follows. DNA carrying the polyhedrin gene is isolated from baculovirus AcNPV and purified. Then Ec
Digestion with oRI restriction endonuclease produces a 7.3 kilobase EcoRI-I fragment containing the polyhedrin gene and other suitable fragments. The above EcoRI-
The I fragment is then cloned into a suitable cloning plasmid Ec
Clone into the oRI site. In this system, the promoter of the polyhedrin gene is present in the nonessential region, and this promoter also functions effectively in the second recombinant vector.

In order to increase the expression level, the polyhedrin gene promoter and the 5'of the polyhedrin gene are used.
It is preferable to use a vector in which a restriction enzyme cleavage sequence is added immediately after the untranslated region, the structural gene sequence of polyhedrin is completely deleted, and the 3'untranslated region of polyhedrin is continued. As such a recombinant vector, pAcY
MS1 (Japanese Patent Laid-Open No. 1-285198) can be mentioned.

Next, a gene for the chimeric protein to be inserted is prepared. The vector used for preparing the gene of the chimeric protein is not particularly limited, but an E. coli vector that can be easily genetically manipulated, such as pBR322 and pBR3.
25, pBR327, pBR328, pUC7, pUC
Examples are plasmids such as 8, pUC9 and pUC19. As long as the genes are linked in the same reading frame so that they are translated as one protein having the antigenic site of both the Japanese encephalitis virus and hepatitis B virus surface antigen proteins, the method for producing the gene encoding the chimeric protein is There is no particular limitation. Usually, a synthetic linker may be used so that the translation stop codon does not exist between the genes encoding the two antigenic proteins.

Then, the gene of the chimeric protein is inserted downstream of the promoter of the first recombinant vector to prepare the second recombinant vector. The insertion method may also be according to a conventional method, for example, the gene fragment of the chimeric protein may be inserted using the restriction enzyme cleavage point artificially added downstream of the promoter of the first vector.
In constructing these first and second recombinant vectors, an Escherichia coli system whose gene can be easily manipulated may be used.

Next, a recombinant virus is constructed. In constructing the recombinant baculovirus, the baculovirus genomic DNA and the second recombinant vector are mixed and then transferred into insect culture cells to cause homologous recombination between the vector DNA and the viral genomic DNA. The construction of the recombinant baculovirus may be carried out according to a conventional method, for example, according to the procedure described below in accordance with the description of Examples in JP-A-60-37988. That is, the second recombinant vector and baculovirus DNA were transfected, and the resulting virus population containing the recombinant baculovirus was recovered, E. Volkman et al. (J. Virol. 19 , 820-832 (19
76)) and subjected to the standard AcNPV polyhedrin plaque assay, plaques that do not form inclusion bodies are selected as recombinant virus candidate strains from the group of developed plaques and the virus is harvested from the plaques. As a method of selecting a virus in which a gene encoding a chimeric protein is integrated from these candidate strains, a plaque is purified using a hybridization method using the gene as a probe.

Insect culture cells are infected with the recombinant virus thus purified. The insect culture cells used here may be those capable of baculovirus growth,
Specific examples thereof include Sf-9 cells derived from Spodoptera frugiperda. Then, it is confirmed whether or not a chimeric protein having the desired antigenicity is expressed in the infected cell lysate. This method may utilize an immunoassay using antiserum or monoclonal antibody against Japanese encephalitis virus or hepatitis B virus. For example, immunoprecipitation (“Antibody”
es a laboratory manual "pp
421-470, (1988), Ed Harlowe.
t. al. Written by Cold Spring Harbor
Laboratory) and the like.

[0017]

EFFECTS OF THE INVENTION According to the present invention, thus, a chimeric protein having antigenic sites of surface antigen proteins of both Japanese encephalitis virus and hepatitis B virus and a recombinant baculovirus expressing the protein can be obtained. This chimeric protein is useful as a bivalent vaccine.

[0018]

EXAMPLES The present invention will be described in more detail with reference to the following examples. Example 1 Preparation of Gene Encoding Chimeric Protein (1) Chimeric Protein R3: S (See FIGS. 1 to 3) DNAs of SEQ ID NOS: 1 and 2 using a DNA synthesizer Genet-II (manufactured by Zeon Corporation) Primers were synthesized and purified. The obtained synthetic primers are respectively P
They were named -R3R (SEQ ID NO: 1) and P-R3L (SEQ ID NO: 2).

SEQ ID NO: 1 and SEQ ID NO: 2 are as follows.

[0020]

[Chemical 1]

[0021]

[Chemical 2]

Using these synthetic primers, a part of the C protein, prM (M) protein, E protein, and part of the NS1 protein of Japanese encephalitis virus disclosed in Japanese Unexamined Patent Publication No. 64-74982 was encoded. .2K
PCR using bp cDNA (203) as a template
Method (Methods in Enzymology, 1
55 , 335-350 (1987), the enzymes used are sold by Takara Shuzo. ) To amplify the gene, pU
It was cloned into C19. The obtained plasmid is pU
It was named CR3. The gene cloned in pUCR3 encodes a part of the surface antigen protein of Japanese encephalitis virus.

Next, the DNA primers shown in SEQ ID NOS: 3 and 4 were synthesized and purified. The obtained synthetic primers were designated as P-HBR (SEQ ID NO: 3) and P-HBL (SEQ ID NO: 4), respectively. Sequence number 3 and sequence number 4 are as follows, respectively.

[0024]

[Chemical 3]

[Chemical 4]

Using these synthetic primers, Nuc
leic Acids Research, 11, 17
The gene was amplified by the same method as described above using the plasmid pBRHBadr containing the DNA encoding the hepatitis B virus surface antigen protein disclosed in 47-1757 (1983) as a template, and cloned into pUC19. The resulting plasmid was named pUCHB. The gene cloned in pUCHB encodes the hepatitis B virus surface antigen protein.

The resulting pUCR3 and pUCHB are
The DNA fragments double-cut with the restriction enzymes BamHI and XhoI respectively were ligated and inserted into the BamHI site of pUC19 to prepare several types of plasmids. By transforming these plasmids into Escherichia coli JM109 and examining the size of the gene fragment cleaved by the restriction enzymes BamHI and XhoI, a part of the surface antigen protein of Japanese encephalitis virus and the surface antigen protein of hepatitis B virus were gened. The plasmid with was selected. The selected plasmid was named pUCR3: S.
The nucleotide sequence of a gene (hereinafter, referred to as R3: S) encoding a chimeric protein contained in this plasmid and the corresponding amino acid sequence (SEQ ID NO: 5) are shown in FIGS.

(2) Chimeric protein R2-3: S (see FIGS. 4 to 8) As a synthetic primer, P-R2R was used instead of P-R3R.
A plasmid (pUCR) having a cDNA encoding a part of the surface antigen protein of Japanese encephalitis virus is prepared by the method described in (1) above except that (SEQ ID NO: 6) is used.
2-3) was prepared. The sequence number 6 is as follows.

[0028]

[Chemical 5]

The subsequent operation is the same as the above (1) except that this plasmid pUCR2-3 is used in place of pUCR3.
A plasmid having several genes prepared by the same procedure as described above, transformed into Escherichia coli in the same manner as in (1) above, and having a gene for a part of the surface antigen protein of Japanese encephalitis virus and the surface antigen protein of hepatitis B virus. Was selected. The selected plasmid was named pUCR2-3: S. The nucleotide sequence of the gene encoding the chimeric protein possessed by this plasmid (hereinafter referred to as R2-3: S) and the corresponding amino acid sequence (SEQ ID NO: 7) are
Shown in FIGS.

(3) Chimeric protein R2: S (Fig. 9-
12) Instead of P-R3L as a synthetic primer, P-R2
A plasmid (pUC having a cDNA encoding a part of the surface antigen protein of Japanese encephalitis virus) was prepared by the method described in (2) above, except that L (SEQ ID NO: 8) was used.
R2) was prepared.

[0031]

[Chemical 6]

In the subsequent procedure, several types of plasmids were prepared by the same procedure as in (1) above, except that this pUCR2 was used in place of pUCR3, and E. coli was transformed in the same manner as in (1) above. A plasmid having a part of the surface antigen protein of Japanese encephalitis virus and the genes for the surface antigen protein of hepatitis B virus was selected. The selected plasmid was named pUCR2: S.
The nucleotide sequence of the gene (hereinafter, referred to as R2: S) encoding the chimeric protein possessed by this plasmid and the corresponding amino acid sequence (SEQ ID NO: 9) are shown in FIGS.
Shown in.

(4) Chimeric protein E291 / 45
1: S (see Figures 13-16) P-291 instead of P-R3R as a synthetic primer
A plasmid (pU having a cDNA encoding a part of the surface antigen protein of Japanese encephalitis virus is prepared by the method described in (1) above, except that R (SEQ ID NO: 10) is used.
CE291 / 451) was prepared. The sequence number 10 is as follows.

[0034]

[Chemical 7]

In the subsequent procedure, several kinds of plasmids were prepared by the same procedure as in the above (1) except that this pUCE291 / 451 was used in place of pUC3 in the plasmid, and Escherichia coli was transformed in the same manner as in the above (1). After conversion, a plasmid having a gene for part of the surface antigen protein of Japanese encephalitis virus and a gene for the surface antigen protein of hepatitis B virus was selected. The selected plasmid was named pUCE291 /
451: It was named S. A gene encoding a chimeric protein possessed by this plasmid (hereinafter referred to as E291 / 45
The base sequence of 1: S and the corresponding amino acid sequence (SEQ ID NO: 11) are shown in FIGS.

Example 2 Preparation of Second Recombinant Vector Inserting Gene Encoding Chimeric Protein As described in Example 2 of JP-A-1-285198, a non-essential DNA region for baculovirus growth was incorporated, Furthermore, a first recombinant vector p having a promoter that functions in baculovirus inserted in its DNA region
AcYMS1 was produced. This pAcYMS1 was cleaved with a restriction enzyme BamHI to prepare plasmids pUCR3: S and pUCR2-3: S prepared in Examples 1 (1) to (4).
Chimeric genes R3: S, R2-3: S, R2: S, and E291 / obtained by treating pUCR2: S and pUCE291 / 451: S with BamHI, respectively.
451: S was inserted using T4 DNA ligase. Each of the obtained plasmids was transformed into Escherichia coli JM109. Plasmids were isolated and purified from the obtained several transformants, and the gene connected to the BamHI site of pAcYMS1 was in the forward direction (the translation direction of the gene was the same as the transcription direction of the polyhedrin promoter). The plasmids connected in the positive direction are selected from the results of the cleavage pattern using an appropriate restriction enzyme. The second recombinant vectors thus obtained were each treated with pAcR
3: S, pAcR2-3: S, pAcR2: S, pAc
It was named E291 / 451: S.

Example 3 Construction of Recombinant Baculovirus Expressing Chimeric Protein F. L. According to the method described in the article by Graham et al. (Virology 52, 1973, 456-467), 2 μg of the second recombinant vector pAcR3: S or p prepared in Example 2 with 1 μg of AcNPV genomic DNA.
AcR2-3: S or pAcR2: S or pAcE
291/451: S (10 μg each), 15 μg
1-HEPES (N-, containing 1 / ml of calf thymus DNA
2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) buffer (pH 7.0) was added to bring the total amount to 95.
The volume was adjusted to 0 μl. While stirring this mixture, 50 ml of 2.5 M CaCl ₂ was added dropwise to precipitate DNA at room temperature. 1 ml of the precipitated DNA was added to S. Frugiperda cells Sf-9 were added to 2 ml of medium containing 4 × 10 ⁶ .

After 4 hours, the cells were washed with a medium, added with 2 ml of a medium containing 10% fetal bovine serum, and incubated at 28 ° C. for 3 days. The medium is then collected with a sterile pipette, which contains recombinant and non-recombinant Ac.
NPV is mixed. To isolate recombinant AcNPV from these mixed populations, the collected medium was appropriately diluted and monolayer-cultured S. Hulgiperda Sf-9 was infected with the diluent and allowed to form plaques. Select a plaque that does not form a virus inclusion body, collect the virus from the plaque, suspend it in PBS (pH 7.0), and partly spot it on a nylon or nitrocellulose membrane for dot hybridization. Is
Again S. Infected Frugiperda and increased the virus.

The spotted membrane was 0.5 N Na
10 minutes with OH, 1M Tris-HCl buffer (pH 7.6)
After treatment with 5M for 3 times, 1.5M NaC was added.
1, treated with 0.5 M Tris-HCl buffer (pH 7.6) for 5 minutes. 2x SSC (1x SSC is 0.15M Na
Cl, 0.015M sodium citrate),
It was baked at 80 ° C. for 2 hours. It was immersed in 6 times SSC, 2% skim milk, 0.1% SDS at 68 ° C. for 2 hours. 6 times S
SC, 2% skim milk, 0.1% SDS, 50 μg /
68 ml containing R3: S gene fragment labeled with ³² P by ml modified salmon sperm DNA and nick translation
Hybridization was performed at 14 ° C for 14 hours.

After washing, the membrane and the X-ray film were overlapped and autoradiography was performed to select spots where the film was blackened. The virus solution corresponding to the blackened spot was diluted with S. Frugiperda cells Sf-9 were infected and plaques appeared. The same operation as described above was performed on the appearing plaques, and the purification operation was repeated until all the appearing plaques were blackened by dot hybridization. The virus thus obtained was the target recombinant baculovirus, and the second recombinant vector used was AcR3: S, A, respectively.
cR2-3: S, AcR2: S, AcE291 / 45
1: S was named.

Example 4 Analysis of Expressed Chimeric Protein (1) Size of Chimeric Protein Produced by Recombinant Baculovirus S. Frugiperda cells (Sf-9) 5 in monolayer
The cells were grown to a density of × 10 ⁶ cells / ml, the growth medium was removed, and 5 p. f. u. The medium containing the recombinant baculovirus AcR2-3: S was added and infected at 28 ° C. The cells were cultured for 3 days after the infection, and the chimeric protein was expressed in the infected cells. The molecular weight of the expressed protein was determined by Western plotting method using a rabbit antiserum against JE-E (Burnet, WN Analyt. Bi.
ochem. 112, 195-203 (1981))
Investigated by. As a result, it was revealed that the AcR2-3: S infected cells produced a protein having a molecular weight of about 52,000 daltons. This molecular weight is the same as that estimated from the amino acid sequence of the chimeric protein.

(2) Size of Chimeric Protein R3: S Produced by Recombinant Baculovirus The recombinant baculovirus used is AcR3: S instead of AcR2-3: S, and the method for confirming the expressed protein is JE-. An immunoprecipitation method using a rabbit antiserum against E (“Antibodies a laboratory”)
y manual "pp421-470 (1988),
Ed Harlow et. al. By Cold Sp
The expressed protein was confirmed by repeating the same operation as in Example 4 (1) above, except that the protein was a ring harbor laboratory. SD of the obtained sediment
Upon S-polyacrylamide gel electrophoresis, two specific bands with molecular weights of 34,000 and 37,000 daltons appeared. This molecular weight was in agreement with the estimated molecular weight of the chimeric protein of 34,000 daltons and the size of the sugar chain attached to 37,000 daltons, confirming that the expected chimeric protein was produced.

(3) Size of Chimeric Protein R2: S Expressed by Recombinant Baculovirus A chimeric protein was expressed in the same manner as in Example 4 (1) except that the recombinant baculovirus used was AcR2: S. , The molecular weight of the expressed protein was investigated. As a result, it was revealed that a protein having a molecular weight of about 40,000 daltons was produced. This molecular weight was the same as that estimated from the amino acid sequence of the chimeric protein.

(4) Size of Chimeric Protein E291 / 435: S Expressed by Recombinant Baculovirus The recombinant baculovirus used was AcE291 / 43.
A chimeric protein was expressed in the same manner as in Example 4 (1) except that the ratio was 5: S, and the molecular weight of the expressed protein was examined. As a result, it was revealed that a protein having a molecular weight of about 36,000 daltons was produced. This molecular weight was the same as that estimated from the amino acid sequence of the chimeric protein.

(5) Reactivity of Chimeric Proteins R3: S and R2-3: S Produced by Recombinant Baculovirus with Antibodies 60 hours after infecting the cells Sf-9 of Frugiperda with the recombinant baculoviruses AcR3: S and AcR2-3: S, 60 hours later, the cells were collected and PBS (pH 7.0) was used.
The cells were washed with PBS, suspended in PBS, spotted on the well of a microslide for fluorescent antibody, and air-dried. This cell
After soaking in acetone at 20 ° C. for 5 minutes and then soaking in PBS containing 2% skim milk for 1 hour to block, each primary antibody was appropriately diluted with PBS containing 2% skim milk (the rabbit antibody shown in Table 1). It was soaked in serum or monoclonal antibody for 1 hour to react. Then wash with PBS, PB
Immerse in secondary antibody diluted with S (fluorescein isothiocyanate-conjugated anti-rabbit IgG or anti-mouse IgG) for 1 hour, and
After washing with BS, a cover glass was placed and the reactivity with the primary antibody was examined with a fluorescence microscope. The results are summarized in Table 1.

[0046]

JE-E used (E of Japanese encephalitis virus
Preparation of a monoclonal antibody against (protein) is described in (J. Virol. 45 , 124-132 (198).
3), J. Gen. Virol. 67 , 2663-26
72 (1986)). The monoclonal antibody 03-6400 against HBsAg is available from Cosmo Bio Inc. (ZYMED Laboratory, Incorporated).
c. The product commercially available from (Manufacturing) was used. As can be seen from Table 1, the chimeric proteins R2-3: S and R
3: S reacted with 4 and 1 of 9 independent monoclonal antibodies against JE-E, respectively. Further, since it also reacted with a monoclonal antibody against HBsAg, it can be seen that the antigenic sites of two viral surface antigen proteins, Japanese encephalitis virus and hepatitis B virus, are formed.

(6) Chimeric proteins R2: S and E291 / 451: S expressed by recombinant baculovirus
Of the recombinant baculovirus used was AcR2: S and AcR2: S instead of AcR3: S and AcR2-3: S.
A chimeric protein was expressed in the same manner as in Example 4 (5) except that it was E291 / 451: S, and the reactivity with the primary antibody was examined. The results are shown in Table 2.

[0049]

As can be seen from Table 2, the chimeric proteins R2: S and E291 / 451: S reacted with the antiserum against JE-E and the monoclonal antibody against HBsAg, and thus were referred to as Japanese encephalitis virus and hepatitis B virus.
It was confirmed that the antigenic sites of two viral surface antigen proteins were formed.

(7) Extracellular Secretion of Chimeric Protein R3: S Produced by Recombinant Baculovirus Frugiperda cells were grown in a monolayer to a density of 5 × 10 ⁶ cells / ml, the growth medium was removed and 5 p. f. u. AcR2-3: S or AcR3:
A medium containing S was added and infection was carried out at 28 ° C. The cells were cultured for 3 days after the infection, and the chimeric protein secreted in the culture supernatant was measured with Auszyme (Dynabot Corporation), which is an HBsAg antigen detection kit. The results are shown in Table 3.
Shown in.

[0052]

As is clear from Table 3, the chimeric protein R3: S produced by AcR3: S was secreted in large amounts in the culture supernatant. It was found that the secreted chimeric protein was formed into particles having a density of about 1.07 by sucrose density gradient centrifugation.

Comparative Example 1 (1) Construction of Recombinant Vaccinia Virus Expressing Chimeric Protein Chimeric proteins R3: S and R prepared in Example 1
2-3: The S gene was designated as pAK8 (JP-A-64-7498
2) was inserted in the BamHI site in the forward direction. Each of the obtained recombinant vectors was designated as pAKR.
It was named 3: S and pAKR2-3: S. These recombinant vectors were transfected to produce recombinant vaccinia virus. As for the method of preparation, except for the transfected plasmid, JP-A-64-7498
The same method as in Example 1 (8) of No. 2 was followed. The DNA probe used for virus purification was the R3: S gene, and the target recombinant vaccinia virus LA
R3: S and LAR2-3: S were obtained.

(2) Extracellular secretion of chimeric proteins R3: S and R2-3: S produced by recombinant vaccinia virus Vero cells were grown in a monolayer to a density of 5 × 10 ⁶ cells / ml and grown. The medium is removed and 5 p.
f. u. The medium containing LAR3: S or LAR2-3: S was added and infected at 37 ° C. The cells were cultured for 2 days after infection, and the secreted chimeric protein was added to the culture supernatant.
It was measured with Auszyme (Dynabot Corporation) which is a BsAg antigen detection kit. The results are shown in Table 4.

[0056]

As is clear from the comparison between Tables 3 and 4, even when the same chimeric protein was expressed, the recombinant baculovirus secreted a much larger amount of protein into the culture supernatant than the recombinant vaccinia virus. This shows the advantage that recombinant baculovirus is easier to produce and purify a chimeric protein.

[Brief description of drawings]

FIG. 1 shows the nucleotide sequence of chimeric protein R3: S and the corresponding amino acid sequence (SEQ ID NO: 5).

FIG. 2 shows the nucleotide sequence of chimeric protein R3: S and the corresponding amino acid sequence (SEQ ID NO: 5).

FIG. 3 shows the nucleotide sequence of chimeric protein R3: S and the corresponding amino acid sequence (SEQ ID NO: 5).

FIG. 4 shows the nucleotide sequence of chimeric protein R2-3: S and the amino acid sequence (SEQ ID NO: 7) corresponding thereto.

FIG. 5 shows the nucleotide sequence of chimeric protein R2-3: S and the amino acid sequence (SEQ ID NO: 7) corresponding thereto.

FIG. 6 shows the nucleotide sequence of chimeric protein R2-3: S and the amino acid sequence (SEQ ID NO: 7) corresponding thereto.

FIG. 7 shows the nucleotide sequence of chimeric protein R2-3: S and the amino acid sequence (SEQ ID NO: 7) corresponding thereto.

FIG. 8 shows the nucleotide sequence of chimeric protein R2: S and the corresponding amino acid sequence (SEQ ID NO: 9).

FIG. 9 shows the nucleotide sequence of chimeric protein R2: S and the corresponding amino acid sequence (SEQ ID NO: 9).

FIG. 10 shows the nucleotide sequence of chimeric protein R2: S and the corresponding amino acid sequence (SEQ ID NO: 9).

FIG. 11 shows the nucleotide sequence of chimeric protein R2: S and the corresponding amino acid sequence (SEQ ID NO: 9).

FIG. 12 shows the nucleotide sequence of chimeric protein R2: S and the corresponding amino acid sequence (SEQ ID NO: 9).

FIG. 13: Base sequence of chimeric protein E291 / 451: S and amino acid sequence corresponding thereto (SEQ ID NO: 1)
1).

FIG. 14: Base sequence of chimeric protein E291 / 451: S and amino acid sequence corresponding thereto (SEQ ID NO: 1)
1).

FIG. 15: Base sequence of chimeric protein E291 / 451: S and amino acid sequence corresponding thereto (SEQ ID NO: 1)
1).

FIG. 16 shows the nucleotide sequence of chimeric protein E291 / 451: S and the corresponding amino acid sequence (SEQ ID NO: 1).
1).

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification number Office reference number FI technical display location A61K 39/295 ADY 9284-4C C12N 15/62 15/86 C12P 21/02 C 8214-4B // (C12N 15/62 C12R 1:92) (C12P 21/02 C12R 1:91) (72) Inventor Koyuki Kamogawa 1-2-1, Yokou, Kawasaki-ku, Kawasaki-shi, Kanagawa Prefecture (72) Kotaro Yasui, Tokyo 2-26-32 Wakabacho, Tachikawa City

Claims (2)

[Claims] 1. A gene encoding a chimeric protein having antigenic sites of surface antigen proteins of both Japanese encephalitis virus and hepatitis B virus is inserted into a nonessential region of the viral genome, and the chimeric protein is introduced into virus-infected cells. A recombinant baculovirus that expresses. 2. A method for producing a chimeric protein having the antigenic sites of surface antigen proteins of Japanese encephalitis virus and hepatitis B virus by infecting insect cells with the recombinant baculovirus according to claim 1.