JPH07265093A - Production of surface antigen protein of japanese encephalitis virus using mammalian cell - Google Patents

Abstract

PURPOSE:To provide a new production process of surface antigen protein of Japanese encephalitis virus which is useful as a prophylactic medicine or diagnostic agent for Japanese encephalitis. CONSTITUTION:An expression vector into which the whole or a part of cDNA coding for the surface antigen protein (E protein) of Japanese encephalitis virus is incorporated together with the whole or a part of cDNA coding for the matrix protein of the virus, or the whole or a part of cDNA coding for the prematrix protein of the virus, or a part of the core protein of the virus, is introduced into the host mammalian cells to effect efficient production of E (envelope) protein.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a Japanese encephalitis virus surface antigen protein useful as a preventive agent or a diagnostic reagent for Japanese encephalitis, and more specifically, a mammal transformed by gene recombination technology. The present invention relates to a method for producing the protein of interest by cells.

[0002]

2. Description of the Related Art Japanese encephalitis is a contagious disease that leaves a high mortality rate and a serious aftereffect caused by infection with Japanese encephalitis virus. Although the number of patients has been decreasing in Japan in recent years, it is still a major epidemic in countries from East Asia to Southeast Asia and even South Asia. Has become a major social problem.

Japanese encephalitis virus is a virus belonging to the genus Flavivirus of the family Togaviridae. The Japanese encephalitis virus gene consists of single-stranded RNA with a molecular weight of approximately 3.8 × 10 ^{6 to} 4.2 × 10 ⁶ , and the structural proteins of Japanese encephalitis virus particles expressed by such genes are classified into the following three types. Separated: E protein occupying the major part of the envelope (surface antigen protein: molecular weight about 53,000), protein M with small envelope (matrix protein: molecular weight about 8,
700) and nucleocapsid protein C (core protein: molecular weight approximately 13,500). Of these, the E protein plays an important role particularly in the establishment of viral infections, so in the fields of preventive medicine and diagnostics, detailed structural analysis of the epitope (antigenic determinant) of the E antigen has been vigorously carried out. . Currently, studies on E antigens are being carried out using the neutralizing activity, hemagglutination activity, erythrocyte hemolytic activity and the like as indicators, and it has been reported that the antigens have regions of at least 9 kinds of epitopes. In addition, it has been revealed that all flaviviruses have similar or similar antigens among species.

[0004] As a preventive drug or vaccine against Japanese encephalitis, a vaccine containing inactivated Japanese encephalitis virus as an active ingredient has been conventionally used. Then, the brain is aseptically collected from the affected mouse, purified and inactivated by the alcohol-protamine method to obtain an inactivated Japanese encephalitis virus vaccine stock solution (National Institute of Preventive Health, `` A Japanese Society of Vaccines '' Revised 2nd Edition (January 2, 1977)
Published by 0 Himaru Zen Co., Ltd.)). However, in such a method for producing a vaccine, a large amount of Japanese encephalitis virus itself is handled, which is extremely dangerous for a person in charge of vaccine production, and the production cost is also high.

As a vaccine, not only the virus itself but also an antigenic protein having the antigenicity of the virus can be used. An attempt to produce a desired antigenic protein in prokaryotic cells or eukaryotic cells by recombinant DNA technology is under consideration. Came to be. The target of this case is the above-mentioned E protein. Attempts were made to express the E protein, which is a Japanese encephalitis virus surface antigen protein, by gene manipulation technology using yeast as a host, but the original quantitative production of E protein was not achieved (34th Japan Virology Society Proceedings, page 91, October 1986).

The present inventors have succeeded in expressing the vaccinia virus, which has been used as a live vaccine, by incorporating a cDNA encoding the E protein of Japanese encephalitis virus in recent years (Japanese Patent Laid-open No. 64-74982). . But,
Although this recombinant virus is promising as a live vaccine, it has a problem in that the production amount is small in order to recover the expressed protein and use it as a vaccine or a diagnostic reagent.
Moreover, contamination of vaccinia virus itself is unavoidable, and side effects such as allergic reactions are feared, and there is a problem in using it as a vaccine as it is.

On the other hand, a method for expressing a foreign gene in insect cells using an insect virus vector has been proposed (JP-A-60-37988 and JP-A-61-578).
No. 7), the amount of expressed protein by these methods is reported to be significantly higher than that of other expression systems.

By the way, Japanese encephalitis virus belonging to the genus Flavivirus of the family Togaviridae has a single-stranded RNA
(The main body that carries the genetic information of the virus), one polypeptide translated from the genome monocistronic in the virus-infected cell is processed into the core protein, matrix protein, E protein and five types of non-constituent proteins. Since it is characterized in that a single polypeptide is cleaved by intracellular proteases to give rise to each protein, it is operative in nature to directly express the E protein-encoding cDNA itself into a baculovirus. It was difficult.

Therefore, when the present inventors selected a region encoding the E protein from the cDNA prepared by using the genomic RNA of Japanese encephalitis virus as a template and incorporating it into a region not essential for the growth of baculovirus, infected insect cells Has completed a technology to express a large amount of Japanese encephalitis virus E protein that can be used as a vaccine or diagnostic reagent (JP-A-1-2).
85198). However, even in this case, when a drug is prepared using the obtained Japanese encephalitis virus E protein as a main component, contamination of insect cell contaminants used for infection is unavoidable, and side effects such as allergic reactions may occur.
There was a problem in using it as a vaccine as it was. In addition, special physical containment facilities for recombinant technology are essential when using these recombinant viruses.

Under the background of such prior art, the inventors of the present invention have made extensive studies aiming at development of a method for producing a large amount of Japanese encephalitis virus E protein, and as a result, prepared using genomic RNA of Japanese encephalitis virus as a template. The region encoding the E protein is selected from the prepared cDNA, and this region is inserted into a suitable expression vector such as pcDL-SRα or pMAM-neo vector, and the vector is used in CHO cells, COS cells and mammalian cells such as Vero. When I moved in,
It was found that the Japanese encephalitis virus E protein, which can be used as a vaccine or a diagnostic reagent, is expressed in large amounts in the host mammalian cells, and the present invention has been completed based on this finding. According to the technology provided by the present invention, a desired E protein is secreted into the culture supernatant of host mammalian cells such as monkey or human cells, the recombinant virus is not contaminated, and the purification is easy and allergy is high. It is possible to prepare a highly safe vaccine with less impurities that cause side effects such as.

[Means for solving problems]

Thus, according to the present invention, a recombinant vector in which a cDNA encoding the E protein of Japanese encephalitis virus is incorporated into a suitable vector is transferred into a host animal cell,
A method for culturing the animal cell and recovering the expressed Japanese encephalitis virus E protein is provided.

The outline of the present invention is as follows. (1) Step of extracting gene RNA of Japanese encephalitis virus: The technique of this step can be carried out by a conventionally known conventional method, for example, a phenol extraction method or the like. (2) Step of preparing double-stranded cDNA complementary to viral RNA: The technique of this step can be carried out by a known conventional method using, for example, reverse transcriptase. (3) Step of cloning cDNA and determining its nucleotide sequence: As a cloning vector used in this step, a plasmid using a prokaryotic cell such as Escherichia coli or Bacillus subtilis as a host, or a bacterio such as λ phage or T4 phage Known ones such as phage-derived vectors can be used. In this step, it is preferable to use a cloning vector and its host cell in an appropriate combination. Specific examples of the vector used for cloning include pBR322, pBR325, pBR32, and the like.
7, pBR328, pUC7, pUC8, pUC9, p
Plasmids such as UC19, phages such as λ phage and M13 phage, or pHC79 (gene, 1
1 , 291, (1980)) and the like. The method for inserting the gene may be in accordance with a conventional method, for example, a Japanese encephalitis virus-derived cD using an artificially added restriction enzyme cleavage point downstream of the vector promoter.
The NA fragment may be inserted. In constructing these vectors, an Escherichia coli system that is easy to genetically manipulate is particularly preferable, and the plasmid vector to be used is not particularly limited as long as it is suitable for the purpose.

(4) Step of identifying that the cloned cDNA contains a gene encoding E protein: A structural gene derived from a cell has a specific DNA base sequence in the initiation and termination regions of its translation. In addition, since the structure of the regulatory gene is similar, the detection and identification of the region of such structural gene is relatively easy. On the other hand, the gene encoding E protein does not have a translation initiation region, a termination region, and a regulatory gene, and a specific nucleotide sequence cannot be adopted as an index. Therefore, the detection and identification of the gene region encoding E protein can be performed. Is extremely difficult. Skilled in rapid nucleotide sequence analysis of cDNA cloned based on the deep insight and excellent technology cultivated by the present inventors, and immunological detection and identification of expression of such cDNA and its expression product The above difficulties can be overcome by using them in combination, and by comparing the nucleotide sequences of the genes encoding the E protein of yellow fever virus and West Nile virus and the amino acid sequence of the E protein that have been reported. It

The cDNA encoding the surface antigen protein of Japanese encephalitis virus can be prepared using, for example, the Nakayama Yoken strain, the JaOAr strain (Yale University Arvovillas Research Unit, Connecticut, USA) or the Sagayama strain (the same place). it can. For example, the cDNA encoding the E protein prepared from the above Sagayama strain is composed of about 1500 base pairs in total as shown in FIGS. 1 to 5, but in the present invention, it is substantially the same as the above cDNA. It may be a modified cDNA (that is, one in which the nucleotide sequence is replaced, inserted or deleted) as long as it has a function. Of course, the amino acid sequences may be modified to different extents as long as they retain substantially the same function.

(5) Step of expressing a region in the gene encoding the cloned E protein: As an expression vector used in this step, a known one such as an expression vector derived from a viral gene such as SV40 can be used. SR that shows high promoter activity over a wide host range
Vectors with α promoter, eg pCDL-S
Rα is a preferred embodiment. The pCDL-SRα vector was created by Dr. Takebe et al.
This is a vector in which the vector primer and linker of the Okayama-Berg method are put together in the same plasmid, and a promoter that exhibits high expression efficiency called SRα is incorporated in place of the SV40 promoter (Molecular and Cellular Biology).
llular Biology), 8 , p. 466-472 (1988)). S
Rα promoter is SV40 promoter and HTLV-
1. LTR R-U5 sequence is connected, and shows high promoter activity in a wide host range.

In this step, it is desirable to select and use an appropriate combination of the expression vector and its host cell. A particularly difficult point to be noted in this step is that a transformant obtained by simply transferring a gene encoding an E protein and a known expression vector into a host cell is immunogenic. It is almost impossible to expect the production of the E protein. That is, in order to link the gene encoding the E protein with a known expression vector, the following measures are required. a) The E protein-encoding gene has no translation initiation and termination regions and is therefore recruited. b) Determine all or part of the gene encoding the E protein linked to the expression vector in order to obtain an expression product with the desired antigenicity and immunogenicity.
c) To enhance the antigenicity and immunogenicity of the expression product. d) Increase the genetic stability of expression vectors and transformants.
e) increase the yield of expression product. f) Secrete the expression product extracellularly to facilitate the purification process. These ideas can be achieved mainly by remodeling and constructing an expression vector.

By the way, the protein of Japanese encephalitis virus is processed into monocistronic as described above, and then processed into surface antigen proteins and the like. Therefore, if the cDNA for the entire viral genome is inserted, it is not necessary to artificially insert the translation start codon and the translation stop codon. The same applies to the case where the cDNA to be inserted has a sequence (eg, ATG) that can serve as a translation initiation codon upstream of the region encoding the E protein. However, when only the cDNA encoding the E protein is to be incorporated, a restriction enzyme cleavage point existing downstream of the promoter is used, and a synthetic linker having a translation initiation codon, a translation termination codon and a restriction enzyme cleavage sequence between the two is used. Will need to be incorporated.

The translation stop codon to be inserted is preferably provided at three positions so that the reading frames of the cDNA encoding the E protein are matched so that they match. The cDNA to be inserted may be one containing a region encoding another protein as long as it contains a region encoding the E protein. For viruses belonging to the genus Flavivirus, a matrix protein (hereinafter referred to as M protein), a prematrix protein (hereinafter referred to as PreM protein), and a core protein (hereinafter referred to as C protein) are coded upstream of the region encoding E protein. However, in the present invention, all or part of the cDNA encoding these proteins may be bound upstream of the cDNA encoding the E protein.

The cDNA to be inserted may be all or part of the cDNA encoding the M protein, all or part of the cDNA encoding the PreM protein, and all of the cDNA encoding the E protein by part of the C protein of Japanese encephalitis virus. Alternatively, a part of them bound upstream is recommended as a preferred embodiment of the present invention. In this case, a large amount of the target E protein is released not only in the cytoplasm but also in the cell culture supernatant, which is a great feature from the viewpoint of subsequent purification of the desired protein.

The host cell into which the E protein expression vector is transferred may be any animal cell capable of expressing the E protein, but preferably the desired transformed animal cell can be easily isolated. Possible mammalian cells such as CHO cells (Chinese hamster ovary cells), COS cells (SV derived from African green monkey kidney)
40 transformed cells) or Vero cells (cells derived from African green monkey kidney) and the like can be used.

In preparing a recombinant transformed animal cell, a gene fragment for transformation is introduced into a host cell by a known method, for example, calcium phosphate method, DEAE-DEX.
It can be performed by the TRAN method, the lipofectin method, the electroporation method, or the like. At this time, in order to facilitate the selection of transformants, a method using animal cells lacking the marker gene on the expression vector is generally used.

(6) Cultivation of transformed cells: Transformed animal cells are cultured under an appropriate growth condition according to a conventional method,
After an appropriate culture time has elapsed, the desired protein is expressed in the cytoplasm of the transformant or in the culture supernatant, and the produced expressed protein of interest is recovered by an appropriate method. Appropriate culture conditions can be easily determined by preliminary experiments.

(7) Step of extraction and purification of expression product: In this step, conventional techniques can be used in combination. For example, the desired protein can be extracted and purified by appropriately combining filtration, salting out, centrifugation, column chromatography and the like. The recovery of the expressed protein from the culture supernatant is not particularly limited as long as it is a conventional biochemical purification method. For example, affinity chromatography using an antibody against Japanese encephalitis virus is a preferred embodiment.

(8) Step of assaying the antigenicity and immunogenicity of the expression product: In this step, conventional techniques can be used in combination. For example, an enzyme-linked immunosorbent assay (ELISA) based on an immunological measurement method and a neutralization test (50% plaque reduction method) can be combined and assayed.

The present invention has been outlined above, and an outline of one embodiment of the present invention is shown in FIG.

Thus, according to the present invention, a cD encoding the E protein derived from Japanese encephalitis virus is contained in a suitable vector.
A recombinant plasmid in which NA is integrated under the control of a promoter is obtained. The recombinant plasmid is allowed to act on a mammalian cell to transform it, and the transformed animal cell is cultured to obtain the E protein of Japanese encephalitis virus. Can be obtained.

The E protein obtained by the present invention is extremely useful as a vaccine or a diagnostic reagent. The vaccine is prepared by adding the antigen of the present invention to a sterilized physiological saline, isotonic solution such as phosphate buffer. In this case, peptone, amino acid, sugar, etc. can be used as a stabilizer. Further, not only a liquid vaccine but also a precipitated vaccine to which an adjuvant is added in order to improve immunogenicity and a freeze-dried vaccine to be highly stabilized and used for transportation can be used. In addition, it is also possible to introduce a saccharide into the protein antigen of the present invention by using a molecular conjugation method or a post-translational modification method in cells to enhance immunogenicity.

Further, the E protein of the present invention is useful as an immunological diagnostic agent for infection of not only Japanese encephalitis virus but also flavivirus genus having similar or similar antigenicity.
For example, it is useful in ELISA, hemagglutination test, hemagglutination inhibition test, passive agglutination test, complement fixation test, and various other tests using antigens or antibodies labeled with fluorescent dyes, enzymes and radioisotopes. .

When used as an antigen for detecting and identifying a Japanese encephalitis virus antibody, it is prepared and used according to the well-known operational methods of the above various test methods. When an antibody is produced using the antigen of the present invention, a test animal is inoculated with the antigen of the present invention to produce the antibody, and then the blood or body fluid of the inoculated animal is collected or a known cell fusion is performed. Use technology.
The polyclonal or monoclonal antibody thus easily prepared can be prepared and used as an antibody for detecting and identifying a Japanese encephalitis virus antigen according to the known procedures of the above various test methods.

Hereinafter, the present invention will be described in detail based on examples, but the present invention is not limited thereto.

[0031]

Example 1 Preparation of cDNA encoding Japanese encephalitis virus E protein (1) Extraction of RNA genome from Japanese encephalitis virus Mosquito-derived cell line C6 / 36 (J. Gen. Virol.
40,531-544 (1978)) in Japanese encephalitis virus S.
After infecting the agayama strain and growing the virus, the culture supernatant and polyethylene glycol were mixed and the Japanese encephalitis virus was purified by centrifugation. Viral genomic RNA (about 11 kbp) was isolated from the purified virus by phenol extraction and ethanol precipitation.

(2) Cloning of cDNA of Japanese encephalitis virus Poly (A) was added to the viral genomic RNA prepared in (1) using poly (A) polymerase (Takara Shuzo), and oligo d was used as a template. Using (T) as a primer, 4 types (A.
First strand cDNA was synthesized by activating reverse transcriptase with the deoxyribonucleotide triphosphate of G.C.T). Next, E. coli RNase H and 4 species (AG
Deoxyribonucleoside triphosphate (C / T) and E. coli D
Double-stranded cDNA was prepared using NA polymerase (Mol
ecular Cloning (Cold Spring Harbor Lab.) p.211-
246 (1982)), a dC chain was added with terminal transferase. On the other hand, after digesting the plasmid pUC9 (Pharmacia) with the restriction enzyme PstI, the dG chain was added with terminal transferase.
The DNAs were mixed and ligated to carry out circularization. This recombinant plasmid was introduced into Escherichia coli HB101 competent cell to obtain a transformant. The plasmid was taken out from the transformant, and the length of the inserted cDNA was compared on an agarose gel.
Sequence analysis on the 5'side for NA (Gene 19 , p. 269)
(1982)). From the result of the sequence analysis, 2
Synthetic oligonucleotide of 0-mer (5'-dATTCCGTACCATG
CAGTCCA-3 ') as a primer and viral genome RN
Using A as a template, prepare cDNA again by the above method,
Ligation into the plasmid pUC9 gave a large number of transformants. From the obtained transformants, transformants containing a recombinant plasmid containing cDNA encoding the target surface antigen protein were selected by the method described below. Regarding the method for preparing a cDNA clone of Japanese encephalitis virus, Japanese encephalitis virus JaOArs 982 strain was used,
An example produced by the same method is Gene 48 , p.195-2.
01 (1986).

(3) Screening of recombinant plasmid pJE4118 containing cDNA (4118) encoding Japanese encephalitis virus surface antigen protein The transformant obtained in (2) was grown on an agar plate,
Replicate to nitrocellulose filter. After gently washing the replicated filter with 0.2% NP-40 (surfactant, Hanai Kagaku), immunoscreening with anti-JE serum yielded one transformant that reacted positively. The plasmid carried by pJE4
It was 118. Then, the plasmid pJE4118 was prepared according to a conventional method (Molecular Cloning p.75-95, described above), and the cDN inserted into pUC9 with the restriction enzyme PstI was prepared.
A was cut out, and using this as a DNA probe, the various transformants obtained in (2) were screened by the colony hybridization method (Molecular Cloning p.382-387, above), and insert c of pJE4118
A plasmid was selected that had a cDNA that overlapped with DNA (4118). The cDNA thus selected (2-2
0) was recovered from the plasmid, and its positional relationship was determined with restriction enzymes (see FIGS. 7 to 8). CDNA (2-20)
Was found to partially overlap with the N-terminus of cDNA (4118).

(4) cDNA encoding a surface antigen protein
Elucidation of nucleotide sequence of A (see FIGS. 1 to 5) Recombinant plasmids pJE4118 and pJE2-20 were digested with restriction enzyme PstI to obtain DNA fragments of about 2.5 kbp and about 1.0 kbp, respectively. For these fragments, the method of Meshing et al. Using M13 phage (Gene 19 , p. 269 (1982)), Science.
(Science) 241 , p.1205 (1981), in the case of cDNA (4118)
In the case of (2-20), sequence analysis was performed from the 3'side.

As a result, the cDN of the overlapping portion
The A sequence was found to be 334 bases from the 3rd codon of the 5th amino acid to the 116th amino acid in FIGS. The amino acid sequence deduced from this base sequence and known amino acid sequences are already known (Science 229 , p.726.
-733 (1985)), the E protein of the Japanese encephalitis virus is shown in FIGS. 1 to 5 by comparison of the amino acid sequences of the E protein of the yellow fever virus (closely related to Japanese encephalitis virus).
It was determined to be from the amino acid to the 500th amino acid.

Similarly, the M protein of Japanese encephalitis virus is from -75th to -1st amino acid shown in FIGS. 1 to 5, and the PreM protein is from -167th to -76th amino acid.
It was judged to be up to th. Furthermore, 5'of PreM protein
The side was judged to encode part of the C protein of Japanese encephalitis virus.

(5) Preparation of cDNA encoding Japanese encephalitis virus surface antigen protein (see FIGS. 1 to 5 and 7 to 8) The amino acid encoded by cDNA (4118) lacks the N-terminal 5 amino acids of E protein. There is. So cDNA (2-
20) was used to join at the position of the AatII site to obtain a cDNA (203) completely covering the E protein. Next, the cDNA (203) was added to HindIII and Eco.
After treatment with RV, a cDNA fragment (J3) of about 2.7 kbp was recovered. This fragment contains the regions encoding the M protein and the PreM protein in addition to the region encoding the E protein.

Example 2 Preparation of Recombinant Vector Inserted with cDNA Encoding Japanese Encephalitis Virus E Protein The cDNA (J3) obtained in Example 1 (5) was transformed into E. coli D.
Repair the ends with NA polymerase (Klenow fragment),
PAcYMS1 and T4DN cut with restriction enzyme SmaI
After ligation with A ligase, JM109 was transformed. Plasmids were purified from several transformants and pA
A plasmid having the cDNA (J3) inserted in the forward direction at the SmaI site of cYMS1 is selected based on the result of the cleavage pattern using an appropriate restriction enzyme. The recombinant vector thus obtained was named pACYMS-J3.

CDNAs encoding the anchor region of C protein (from arginine at 105th position to alanine at 127th position), Pre-M, M protein and E protein were designated as pcDL-
It was inserted into the PstI-EcoRI site of the SRα296 vector (pcDL-SRα-J12). This recombinant was introduced into E. coli DH1 strain, transformed and cloned.

Similarly, all C proteins, Pre-M, M
CDNA encoding the protein and E protein was labeled with pcDL
-SRα296 vector was inserted into the PstI-EcoRI site, and this recombinant was introduced into Escherichia coli DH1 strain for transformation and cloning (pcDL-SRα-J1).
8).

For control, cDNAs encoding NS2B and NS3, which are proteins of the non-structural region of Japanese encephalitis virus
Was inserted into the pcDL-SRα296 vector, and pcDL-
SRα-JNS2B3.

Example 3 Expression of Japanese Encephalitis Virus E Protein in Mammalian Cells as Host (1) Transformation Method of Animal Cells The various recombinant vectors obtained in Example 2 were subjected to lipofectin method to COS7 cells (African green monkey). Kidney-derived SV
40 transformed cells). A DNA amount of 100 ng / 6 cm plate was used. 5 μg / in Vero cells
Using the plate, it was possible to obtain an expression level close to that of COS cells, although there was no increase in copy number due to replication. As other cells, all cells such as human and monkey are considered to be usable.
The pMAM-neo vector (using CHO cells) can be considered as another usable vector.

(2) Evaluation system Immunoprecipitation: 2 days after transfection, ³⁵ S-methionine and ³⁵ S-cysteine (0.4 mCi /
Cells were labeled with 6 cm) for 16 hours. Collect the supernatant,
The cells are washed and then lysed in RIPA solution, and each is treated with anti-JE
Immunoprecipitation was performed using V antibody and protein A-Sepharose, and viral proteins were analyzed by 16% SDS-PAGE. In addition, the cell supernatant was collected and the viral protein was analyzed by Western-blotting. The results are shown in Fig. 9. When each expression vector in which the E protein coding region was inserted was used, expression of the desired E protein was confirmed in the cytoplasm, but in the vector without the E protein coding region (pcDL-SRα-JNS2B3) As expected, E protein expression was not observed. In the case of pcDL-SRα-J12, a vector in which the cDNAs encoding the anchor region of C protein (from arginine at position 105 to 127 at alanine), Pre-M, M protein and E protein were inserted, not only cytoplasm It is noteworthy that E protein is also produced in the culture cell supernatant.

Analysis using fluorescent antibody: COS cells were grown on a chamber slide, and similarly pcDL-SRα-J
12 were transfected. Two days later, the cells were fixed with acetone and immunostained with anti-JEV antibody and anti-mouse IgG-FITC as a secondary antibody. In FIG. 10, a white portion shows a portion where the expressed substance and the antibody react with each other. In the case of pcDL-SRα-J12, efficient expression of E protein is observed (see FIG. 10).

ELISA: The culture supernatant of COS7 cells transfected with pcDL-SRα-J12 was recovered and treated with 10% PEG, 1M NaCl to remove E protein and P
ReM protein is precipitated, and the precipitation is adjusted to 10-40% suc
Fractionated with rose gradient and the resulting E protein was electrophoresed using anti-E protein monoclonal antibody (201).
It was detected by the ISA method. The peak fractions were collected and the epitope was detected with anti-JEV antibody or various anti-E protein monoclonal antibodies. The ELISA method uses anti-mouse IgG-Peroxidase as the secondary antibody and
Color was developed with a D, H ₂ O ₂ color-developing system, and the absorbance was measured at OD ₄₉₂ . The antibodies used are described in J. of Virology, 45 , p.
124-132 (1983).

[0046]

[Sequence list]

 SEQ ID NO: 1 Sequence Length: 2910 Sequence Type: Nucleic Acid Number of Strands: Single Strand Topology: Linear Sequence Type: cDNA Origin Biological Name: Virus Sequence ATCACGTTCT TCAAGTTTAC AGCATTAGCC CCGACCAAGG CGCTTTTAGG CCGATGGAAA 60 GCAGTGGAAA AGAGTGTGGC AATGAAACAT CTGATAGTT TCAA ACTTGGAACA 120 CTCATTGACG CCGTGAACAA GCGGGGCAGA AAACAAAACA AAAGAGGAGG AAATGAAGGC 180 TCAATCATGT GGCTCGCAAG CTTGGCAGTT GTCATAGCTT ACGCAGGAGC AATGAAGTTG 240 TCGAATTTCC AGGGGAAGCT TTTGATGACC ATCAACAACA CGGACATTGC AGACGTTATC 300 CTGATTCCCA CCTCAAAAGG AGCGAACATA TGCTGGGTCC GGGCAATAGA CGTCGGCTAC 360 ATGTGTGAGG ACACTATCAC GTACGAATGT CCTAAGTTCA CCATGGGCAA TGATCCAGAG 420 GATGTGGATT GTCGGTGTGA CAACCAAGAA GTCTACGTCA AATATGGACG GTGCACGCGG 480 ACCAGGCATT CCAAGCGAAG CAGGAGATCC GTGTCGGTCC AAACACATGG GGAGAGTTCA 540 CTAGTGAATA AAAAAGAGGC TTGGCTGGAT TCAACGAAAG CCACACGGTA TCTCATGAAA 600 ACTGAGAACT GGATCATAAG GAATCCTGGC TATGCTTCTC TGGCGGCGGT ACTTGGCTGG 660 ATGCTTGGCA GTAACAACGG TCAACGCGTG GTATTT ACCA TCCTCCTGCT GTTGGTCGCT 720 CCGGCTTACA GTTTTAATTG TCTGGGAATG GGCAATCGTG ACTTCATAGA AGGAGCCAGT 780 GGAGCCACTT GGGTGGACTT GGTGCTAGAA GGAGATAGCT GCTTGACAAT CATGGCAAAC 840 GACAAACCAA CATTGGACGT CCGCATGATT AACATCGAAG CTAGCCAACT TGCTGAGGTC 900 AGAAGTTACT GCTATCATGC TTCAGTCACT GACATCACGA CGGTGGCTCG GAGCCCCACG 960 ACTGGAGAAG CCCACAACGA GAAGCGAGCT GATAGTAGCT ATGTGTGCAA ACAAGGCTTC 1020 ACTGATCGTG GGTGGGGCAA CGGATGTGGA CTTTTCGGGA AGGGAAGCAT TGACACATGT 1080 GCAAAATTCT CCTGCACCAG CAAAGCGATT GGAAGAACAA TCCAGCCAGA AAACATCAAA 1140 TACGAAGTTG GCATTTTTGT GCATGGAACC ACCACTTCGG AAAACCATGG GAATTATTCA 1200 GCGCAAGTTG GGGCGTCCCA GGCGGCAAAG TTCACAGTAA CACCCAATGC TCCTTCGATA 1260 ACCCTCAAAC TTGGTGACTA CGGAGAAGTC ACGCTGGACT GTGAGCCAAG GAGTGGACTG 1320 AACACTGAAG CGTTTTACGT CATGACCGTG GGGTCAAAGT CATTTCTGGT CCATAGGGAA 1380 TGGTTTCATG ACCTCGCTCT CCCCTGGACG TCCCCTTCGA GCACAGCGTG GAGAAACAGA 1440 GAACTCCTCA TGGAATTTGA AGAGGCGCAC GCCACAAAAC AGTCCGCTGT TGCTCTTGGG 1500 TCACAGGAAG GAGGCCTCCA TCAGGCGTTG GCAGGAGCCA TCGTGG TGGA GTACTCAAGC 1560 TCAGTGAAGT TAACATCAGG CCACCTGAAA TGTAGGCTGA AAATGGACCC CCTGAAGTTG 1620 AAAGGCACTA CGTACGGCAT GTGTACAGAA AAATTCTCGT TCGCGAAAAA TTCGGCAGAC 1680 ACTGGCCACG GAACAGTCGT CATTGAACTA TCCTACTCTG GGAGTGATGG CCCCTGCAAA 1740 ATTCCAATTG TCTCCGTTGC GAGCCTCAAT GACATGACCC TGGTTGGCCG GCTGGTGACA 1800 GTGAACCCTT TCTGCGCGAC TTCCAGTGCC AACTCAAAGG TGCTGGTCGA GATGGAACCC 1860 CCCTTCGGAG ACTCCTACAT CGTGGTTGGA TGGGGAGACA AGCAGATCAA CCACCATTGG 1920 CACAAAGCTG GAAGCAGCGC TAGCAAGGCC TTTTCAACAA CTTTGAAGGG AGCTCAAAGA 1980 CTGGCAGCGT TGGGCGACAC AGCCTGGGAC TTTGGCTCCA TTGGAGGGGT CTTCAACTCC 2040 ATAGGAAAAG CCGTTCACCA AGTGTTTGGT GGTGCCTTCA GAACACTCTT TGGGGGAATG 2100 TCTTGGATCA CACAAGGGCT AATGGGTGCC CTACTACTCT GGATGGGCGT CAACGCACGA 2160 GACCGATCAA TTGCTTTGGC CTTCTTAGCC ACAGGAGGTG TGCTCGTGTT CTTAGCGACC 2220 AATGTGCATG CTGACACTGG ATGTGCCTTT GACATCACAA GAAAAGAGAT GAGATGTGGA 2280 AGTGGCATCT TTGTGCACAA CGACGTGGAA GCCTGGGTGG ACAGGTATAA ATACTTGCCA 2340 GAAACGCCCA GATCCCTAGC GAAGATCGTC CACAAAGCGC ACAAGGAAGG C GTGTGCGGA 2400 GTCAGATCTG TCACTAGATT GGAGCACCAA ATGTGGGAAG CCGTACGGGA CGAATTGAAC 2460 GTCCTGCTCA AAGAGAATGC AGTGGACCTC AGTGTGGTTG TGAACAAGCC CGTGGGAAGA 2520 TATCGCTCAG CCCCTAAACG CCTATCCATG ACGCAAGAGA AGTTTGAAAT GGGCTGGAAA 2580 GCATGGGGGA AAAGCATTCT CTTTGCCCCG GAATTGGCTA ACTCCACATT TGTCGTAGAT 2640 GGACCTGAGA CAAAGGAATG CCCTGATGAG AATAGAGCTT GGAACAGCAT GCAAATCGAA 2700 GACTTCGGCT TTGGCATCAC ATCAACCCGT GTGTGGCTGA AAATTAGAGA GGAGAGCACT 2760 GACGAGTGTG ATGGAGCGAT CATAGGCACG GCTGTCAAAG GACATGTGGC AGTCCATAGT 2820 GACTTGTCGT ACTGGATTGA GAGTCGCTAC AACGACACAT GGAAACTTGA GAGGGCAGTC 2880 TTTGGAGAGG TCAAATCTTG CACTTGGCCA 2910

[Brief description of drawings]

FIG. 1 is a diagram showing the nucleotide sequence and amino acid sequence of a cDNA containing a region encoding the Japanese encephalitis virus E protein.

FIG. 2 is a diagram showing the nucleotide sequence and amino acid sequence of a cDNA containing a region encoding the Japanese encephalitis virus E protein. (Continued from Figure 1)

FIG. 3 is a diagram showing the nucleotide sequence and amino acid sequence of a cDNA containing a region encoding the Japanese encephalitis virus E protein. (Continued from Figure 2)

FIG. 4 is a diagram showing the nucleotide sequence and amino acid sequence of a cDNA containing a region encoding the Japanese encephalitis virus E protein. (Continued from Fig. 3)

FIG. 5 is a diagram showing the nucleotide sequence and amino acid sequence of a cDNA containing a region encoding the Japanese encephalitis virus E protein. (Continued from Fig. 4)

FIG. 6 is a diagram showing an outline of an operation procedure of the present invention.

FIG. 7 is a diagram showing the positional relationship of restriction enzyme sites of cDNA and the position of gene fragments according to the present invention.

FIG. 8 is a diagram showing the positional relationship of restriction enzyme sites of cDNA and the position of gene fragments according to the present invention. (Continued from Fig. 7)

FIG. 9 is an electrophoretogram (drawing-substituting photograph) showing the result of immunoprecipitation of the protein expressed by the present invention.

FIG. 10 is a morphological diagram (drawing-substituting photograph) of an organism (cell) showing an analysis result of a protein expressed by the present invention using a fluorescent antibody.

Continuation of front page (51) Int.Cl. ⁶ Identification number Office reference number FI Technical display location C12N 15/09 ZNA // A61K 39/12 ADY (C12P 21/02 C12R 1:91) (C12N 5/10 C12R (1:91) (C12N 5/00 B C12R 1:91)

Claims (6)

[Claims] 1. Japanese encephalitis expressed by transferring a recombinant expression vector incorporating all or part of a cDNA encoding a Japanese encephalitis virus surface antigen protein (E protein) into mammalian cells, culturing the animal cells, and expressing the cells. Virus surface antigen
A method for producing a Japanese encephalitis virus surface antigen protein (E protein), which comprises recovering (E protein). 2. A cDNA encoding a Japanese encephalitis virus matrix protein, wherein all or part of a cDNA encoding a Japanese encephalitis virus surface antigen protein (E protein).
The method for producing a Japanese encephalitis virus surface antigen protein (E protein) according to claim 1, which is incorporated together with all or a part thereof. 3. A cDNA encoding the Japanese encephalitis virus pre-matrix protein, wherein all or part of the cDNA encoding the Japanese encephalitis virus surface antigen protein (E protein).
The method for producing a Japanese encephalitis virus surface antigen protein (E protein) according to claim 1, which is incorporated with all or part of NA. 4. A cDNA encoding the Japanese encephalitis virus matrix protein, wherein all or part of the cDNA encoding the Japanese encephalitis virus surface antigen protein (E protein).
4. The surface of the Japanese encephalitis virus according to claim 1, which is incorporated together with all or part of the cDNA, all or part of the cDNA encoding the pre-matrix protein of Japanese encephalitis virus, and part of the core protein of Japanese encephalitis virus. A method for producing an antigen protein (E protein). 5. The Japanese encephalitis virus surface antigen protein (E protein) according to claim 4, wherein a part of the core protein of the Japanese encephalitis virus is the anchor region (105th arginine to 127th alanine) of the core protein. Production method. 6. The mammalian cells are CHO cells (Chinese hamster ovary cells), COS cells (African green monkey kidney-derived SV40 transformed cells) and Vero.
The method for producing a Japanese encephalitis virus surface antigen protein (E protein) according to claim 1, which is selected from cells (cells derived from African green monkey kidney).