JPH0585943A - Manifestation of non-a, non-b hepatitis virus gene using recombinant vaccinia virus and non-a non-b hepatitis vaccine - Google Patents

Abstract

PURPOSE:To prepare a vaccine for preventing or improving non-A, non-B hepatitis by manifesting a non-A, non-B hepatitis virus antigen protein by using a recombinant vaccinia virus and using the resultant antigen protein. CONSTITUTION:By homologeous recombination between a gene containing a base sequence coding a non-A, non-B hepatitis virus antigen protein (preferably envelope glucoprotein), a plasmid containing a virus promoter capable of manifesting the above-mentioned gene and a vaccinia virus gene (preferably hemagglutinin gene) not essential for propergation of a vaccinia virus, preferably plasmid P7.5-E12 and the vaccinia virus, a recombinant vaccinia virus (preferably P7.5-E12/RLV) containing an inserted gene coding the non-A, non-B hepatitis virus antigen protein is obtained. The objective vaccine is prepared by using the antigen protein manifested by using the above-mentioned virus or an attenuated or inactivated recombinant vaccinia virus.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to the expression of a non-A non-B hepatitis virus antigen protein using a recombinant vaccinia virus and its application to a vaccine.

[0002]

BACKGROUND OF THE INVENTION Non-A non-B hepatitis is a viral infectious hepatitis, and after its onset, many become chronic, and cirrhosis and liver cancer shift to a high rate, which is a major social problem. Preventive measures are needed. There are two types of prevention of viral diseases: the method using a vaccine and grasping the infection situation and cutting off the path. Of these, the diagnostic kit required to understand the infection status is M. Ho from Chiron.
It has already been developed by ughton et al. (Japanese Patent Publication No. 2-500880) or Maki et al. (Japanese Patent Application No. 2-180889), and infection by blood transfusion can be prevented at a fairly high rate. However, about half of non-A non-B hepatitis patients (about 500,000 /
Years) are non-transfusion-infected persons, there is currently no effective means to prevent these infections, and the development of preventive vaccines is desired.

Already with regard to this type of vaccine, M.
Houghton et al., In the above publication, disclose the human hepatitis C virus (H
CV) vaccines containing immunogenic polypeptides containing epitopes, vaccines containing inactivated HCV, and vaccines containing attenuated HCV are disclosed.

[0004]

SUMMARY OF THE INVENTION It is an object of the usually often virus itself inactivated by drugs formalin or the like is used in the vaccine, non-A, non-B hepatitis virus has not been yet isolated, also in Since there is no virus growth system in vitro , the virus cannot be obtained in large quantities. Therefore, it has been suggested to express a non-A non-B hepatitis virus gene in Escherichia coli, yeast, animal cells and the like to obtain a vaccine antigen (Japanese Patent Publication No. 2-500880).

In general, a protein having a sugar such as a virus envelope protein has a high immunogenicity and is excellent in immunization as compared with a protein having no sugar, but an effective method for expressing such a glycoprotein and For that acquisition, M. Hough
ton et al. did not teach at all. An expression system using animal cells is considered to express a glycoprotein such as a virus envelope protein in a form in which a sugar chain is added. However, in an expression system using animal cells, the expressed protein is generally obtained only with an efficiency of several mg / l, and it is quite difficult to raise the purity to a degree of purification that can be used as a vaccine.

As a result of earnest research, the present inventors have found that a vaccinia virus is recombined with a recombinant plasmid containing a gene containing a nucleotide sequence encoding a non-A non-B hepatitis virus antigen protein in the vaccinia virus gene and a virus promoter. It was found that a non-A non-B hepatitis B virus antigen protein having a sugar can be expressed by using a recombinant vaccinia virus obtained by homologous recombination between the present invention, and completed the present invention.

It is an object of the present invention to provide a non-A non-B hepatitis virus antigen protein expression system using a recombinant vaccinia virus.

Another object of the present invention is to provide a naturally occurring or near-naturally occurring antigenic protein obtained through this expression system.

The present invention is further directed to the application of the attenuated or inactivated recombinant vaccinia virus and the antigenic protein to vaccines.

When the recombinant vaccinia virus is used as a vaccine, it can be used in a very small amount because the virus gives immunity by growing at the inoculation site, and the purification is easy.

[0011]

The present invention is not essential for the growth of vaccinia virus, a gene containing a nucleotide sequence encoding a non-A non-B hepatitis virus antigen protein, a viral promoter capable of expressing the gene, and vaccinia virus. Provided is a plasmid containing a vaccinia virus gene.

The plasmid is an essential component for producing a recombinant vaccinia virus by homologous recombination with vaccinia virus.

In a gene containing a nucleotide sequence encoding a non-A non-B hepatitis virus antigen protein, the encoded antigen protein may be either a non-A non-B hepatitis virus non-structural protein or a structural protein. In order to achieve effective immunization, structural proteins, particularly proteins constituting the envelope and core are preferred. The envelope is a structure constituting an animal virus coat, and includes a lipid bilayer membrane similar to a cytoplasmic membrane and a glycoprotein structure made by a viral gene. This glycoprotein structure has one end embedded in the membrane and the other end protruding to the outside in the form of a protrusion (called a spike). The core, also called the internal capsid, constitutes a shell made of proteins that wrap the viral genome. Although the antigen protein does not necessarily require sugar, it is preferably a glycoprotein in order to enhance immunogenicity, and in this case, the characteristics of the present invention that enable efficient expression of the glycoprotein are sufficiently exerted. obtain. In the examples described below, the gene encoding the envelope antigen glycoprotein gp35 of non-A non-B hepatitis virus was actually incorporated into a plasmid to realize its expression.

The viral promoter may be of any type, so long as it can express a gene containing the nucleotide sequence encoding the antigen protein. As such a promoter, a vaccinia virus-derived promoter (7.
5 kD protein, ATI promoter), cytomegalovirus promoter, etc., but a vaccinia virus-derived promoter is preferable for effective expression of the gene in the recombinant vaccinia virus.

The vaccinia virus gene is preferably a gene other than the gene related to the growth system of this virus, and the hemagglutinin (HA) gene can be particularly preferably used from the viewpoint of screening for recombinant virus.

The arrangement of these genes and promoters is generally such that both genes are vaccinia virus genes so that the gene containing the nucleotide sequence encoding the non-A non-B hepatitis virus antigen protein is located downstream of the viral promoter. It is suitable to take the form inserted inside.

According to an embodiment of the invention plasmid P7.
5-E12 can be preferably used as the plasmid of the present invention,
This particular plasmid is also included within the scope of the invention. As shown in the Examples below, the plasmid of this example was prepared by first isolating the HA gene from the vaccinia virus WR strain, and
By cloning into UC18, inserting a 7.5 kD protein promoter derived from vaccinia virus into the NruI site of this HA gene, and inserting a gene containing a nucleotide sequence encoding a non-A non-B hepatitis virus antigen protein downstream thereof. It can be produced (see FIG. 1).

The present invention also provides a recombinant vaccinia virus obtained by homologous recombination of the above-mentioned recombinant plasmid and vaccinia virus.

The recombinant vaccinia virus of the present invention comprises the steps of linearly restricting the above recombinant plasmid of the present invention, transfecting an animal cell infected with vaccinia virus to carry out homologous recombination, and It can be produced by a method including a step of screening a recombinant virus having a gene encoding a non-B hepatitis A virus antigen protein inserted therein and recovering it. This manufacturing method is also included in the scope of the present invention.

According to the embodiment of the present invention, a vaccinia virus lister strain can be preferably used as the vaccinia virus. Mammalian cells are suitable as animal cells for infecting the virus, and examples thereof include rabbit kidney passage cells (RK-13 cells).

Recombinant vaccinia virus P7.5-E12 / RLV according to an embodiment of the invention may preferably be used,
This is also included within the scope of the present invention. In this recombinant virus, the linearized plasmid p7.5-E1
2 / RLV is inserted almost at the center of the gene encoding the HA (hemagglutinin) protein in the vaccinia virus gene. Therefore, normal hemagglutinin protein is not produced. In addition, since the inserted non-A non-B hepatitis virus gene has a start codon ATG at the head, mRNA is newly synthesized from this and protein synthesis is performed.

The screening of recombinant vaccinia virus is carried out by the erythrocyte adsorption test (HA test) (Shida, H., v
irology 150: 451-462, 1986) to obtain a recombinant virus candidate strain of HA (-), and then plaque hybridization using a nucleotide sequence encoding a peptide constituting a non-A non-B hepatitis virus antigen protein as a probe. Can be carried out by a method of detecting a positive clone by.

The present invention further comprises a gene containing a nucleotide sequence encoding a non-A non-B hepatitis virus antigen protein by infecting an animal cell with the recombinant vaccinia virus produced as described above and culturing the cell. And a method for producing an expressed non-A non-B hepatitis virus antigen protein, which comprises recovering the expressed protein.

Mammalian cells are suitable as the animal cells, and in particular, rabbit kidney passage cells (RK-13 cells) can be preferably used. In addition, the culture conditions are determined depending on the animal cells used, and a medium capable of growing, a culture temperature, a culture time, etc. are appropriately selected. For example, in the case of P7.5-E12 / RLV described above, bovine serum-containing EMEM medium containing fructose instead of glucose may be used.

Expression of the non-A non-B hepatitis virus antigen protein can be carried out by conventional methods such as immunoprecipitation and indirect fluorescent antibody method using an antibody against the antigen protein or non-A non-B hepatitis patient serum containing the antibody. It can be confirmed by immunological techniques (Figures 2 and 3).

The present invention also provides an expressed non-A non-B hepatitis virus antigen protein having a sugar among the non-A non-B hepatitis virus antigen proteins thus expressed.

Expression of glycoproteins has never been realized in the non-A non-B hepatitis virus-related field,
The expression of the gene was made possible for the first time by the invention of an expression system using a recombinant vaccinia virus containing a gene containing a nucleotide sequence encoding the antigenic glycoprotein.

As used herein, the expression "expressed non-A non-B hepatitis B virus antigen protein having a sugar" is meant to include both natural non-A non-B hepatitis B virus antigen glycoprotein and its analogs. To do. In addition, the analogue of the native antigen glycoprotein is capable of blocking the entry of non-A non-B hepatitis virus into the animal body or eradicating the virus infecting animals. It means a range of variants that can produce antibodies against hepatitis B virus.

The present invention further provides a vaccine for treating or preventing non-A non-B hepatitis, which comprises the expressed non-A non-B hepatitis virus antigen protein having the above sugar.

The present invention also provides a vaccine for treating or preventing non-A non-B hepatitis, which comprises an attenuated or inactivated recombinant vaccinia virus.

Vaccinia virus is a member of the genus Poxvirus used to generate human smallpox immunity. After the recombinant vaccinia virus is propagated in animal cells, tissues, organs, etc., it can be attenuated in pathogenicity to give a live attenuated vaccine, or can be inactivated with formaldehyde to give an inactivated vaccine. In Example 7, which will be described later, inoculation of the recombinant vaccinia virus P7.5-E12 / RLV into the dorsal skin of a rabbit actually produces an antibody against the non-A non-B hepatitis virus antigen protein in rabbit serum. This is demonstrated (Fig. 4).

The vaccines according to the invention are generally such that the immunogen is contained in a water-in-oil emulsion or adsorbed on an inorganic gel such as aluminum hydroxide, aluminum phosphate or comprises a conventional organic adjuvant. It is used in the form of an adjuvant vaccine or a solution in a liquid solvent such as physiological saline or glycerol, but is not limited thereto. In addition, inoculation is preferable as a method of applying the vaccine, and the vaccine is administered in an amount that achieves the desired preventive or therapeutic effect, and the amount depends on the age, weight, symptoms, etc. of the individual.

[0033]

The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples unless the gist of the present invention is changed.

Example 1 Expression of hemagglutinin (HA) gene of vaccinia virus
Cloning vaccinia virus WR strain with sucrose density gradient centrifugation [Jo
klik.WK, Virology, 18 , 9-18 (1962)], 50m
Suspend in M Tris-HCl (pH 7.4) buffer (1 mM EDTA, containing 0.5% sodium dodecyl sulfate), add proteinase K to a concentration of 250 to 1000 μg / ml, and add 37
After incubating at 0 ° C. overnight, it was extracted with a phenol: chloroform (1: 1) solution under saturation with TE buffer three times, and ethanol precipitation was performed to obtain a viral DNA. This DNA was digested with HindIII in medium salt buffer,
About 50 kb of Hind I by agarose gel electrophoresis
The IIA fragment was obtained. This Hind IIIA fragment was digested with SalI in a high salt buffer to give 3'of the Hind IIIA fragment.
The approximately 1.8 kbp HindIII-SalI fragment located at the end was isolated by agarose gel electrophoresis.

On the other hand, the plasmid pUC13 was linearized by digestion with HindIII in a medium salt buffer and subsequently with SalI in a high salt buffer. This linearized plasmid was isolated by agarose gel electrophoresis.

The above HindIII-SalI fragment and the linearized plasmid were ligated with T4 ligase in ligation buffer, and this reaction mixture was used to E. coli JM103.
Was transformed. The plasmid was recovered from each transformant by the alkali extraction method. Further analysis with restriction enzymes was performed to select a plasmid having the desired gene constitution, which was designated as pHA13.

Example 2 Vaccinia virus 7.5 kD protein promoter
Preparation of multi-vector for recombination using the vector A recombinant vector for incorporating a 7.5 kD protein promoter of vaccinia virus and a foreign gene linked to the downstream thereof into the genomic HA gene of vaccinia virus was prepared by the following method.

First, DN from vaccinia virus WR strain
A was extracted in the same manner as in Example 1 and Venkatesan
Et al. [Venkatesan & B. Moss, J. Virol. 33 , 738-
745 (1981)], the 7.5 kD protein promoter was isolated. That is, the extracted WR strain DNA was
After digesting with I, the resulting 0.9 kbp DNA fragment was digested with p
It was cloned into the SalI site of UC18. further,
6. digest this plasmid with RsaI and HincII,
0.26 kbp RsaI containing a 5 kD protein promoter
-HincII fragment was obtained. This DNA fragment was cloned into pUC18 (p7.5-18).

Next, 7.5k isolated by the above method
The D protein promoter was inserted into the NruI site of the HA gene cloned in Example 1 by the following method.

The plasmid p 7.5-18 was digested with EcoRI and HindIII in a medium salt buffer to excise the 7.5 k promoter gene of about 0.29 kbp and isolated by agarose gel electrophoresis. The isolated gene fragment is dT in nick translation buffer.
The reaction was performed with Klenow fragment in the presence of TP, dCTP, dATP and dGTP to make the ends blunt.

On the other hand, the plasmid pHA-13 was linearized by digesting it in NruI buffer. The linearized plasmid and the blunted gene fragment were ligated with T4 ligase in a ligation buffer to transform Escherichia coli JM109. As a result of analysis with a restriction enzyme, the plasmid obtained from the transformant had a 7.5 kD promoter (P7.5
) Was inserted in the same direction as the HA gene. This was named pVR-1 (Fig. 1).

Example 3 Preparation of Recombinant Plasmid A recombinant vector used for integrating the gene of the envelope region of non-A non-B hepatitis virus into the HA gene of the vaccinia virus genome was prepared by the following method.

Clone C10-E12 having a region encoding a structural protein of non-A non-B hepatitis virus
3444) (Japanese Patent Application No. 2-413844) was digested with KpnI in a low salt buffer, and a gene fragment of about 1.0 kbp was isolated by agarose gel electrophoresis to obtain a gene fragment (FIG. 1).

The recombinant plasmid pVR-1 was linearized by similarly digesting it with KpnI in a low salt buffer. Both were ligated with T4 ligase in ligation buffer. Escherichia coli HB101 was transformed with this ligation product, and the plasmid was recovered from the transformant by the alkaline method. Analysis with restriction enzymes was carried out to obtain a plasmid in which the envelope region was ligated in the same direction downstream of the promoter, which was designated as P7.5-E12.

Example 4 Preparation of recombinant vaccinia virus a) Preparation of DNA to be used for transfection 20 using DIAGEN plasmid kit (manufactured by DIAGEN)
150 µg of plasmid P7.5-E12 was extracted from E. coli cultured in 0 ml of culture medium. This plasmid is CsC
It was purified by a density gradient centrifugation method. That is, the plasmid was dissolved in a CsCl solution (containing EtBr) of ρ = 1.47 g / ml and filled in a quick seal tube (Beckman Co., Ltd., Ultra Clear, 13 × 51 mm).
It was centrifuged at 55,000 rpm for 15 hours. (VTi 65.2 rotor, Beckman ultracentrifuge). After centrifugation, closed circular
The band of the plasmid DNA was collected and extracted with isopropanol three times to remove EtBr. Then, a purified plasmid was obtained by ethanol precipitation.

Purified P7.5 above in medium salt buffer
-E12 was cleaved by HindIII. 25 μg of this linearized recombinant vector was prepared for transfection.

B) Transfection Gene transfer was carried out by the electroporation method of Perkes et al. [Marion E. Perkus, Keith Limbach and Enzo Paolett.
i, J. Virol. 63 , 3829-3836 (1989)]. That is, the rabbit kidney-derived cell line RK13 cells monolayer-cultured in a culture bottle of 175 cm ² were infected with vaccinia virus Lister strain at moi 5, and
After adsorbing under 5% CO ₂ for 1 hour, infected cells were collected using trypsin. The collected cells were washed twice with HeBS buffer (pH 7.05), and 0.8 ml of H 2 was added together with 25 μg of DNA prepared for transfection in a).
Suspended in eBS buffer. Transfer the cell suspension to a pulsar cuvette (manufactured by Bio-Rad) and in this state 10
Cooled on ice for 1 min. After this, a pulse of 200 V (Capacitance, 960 μF) was applied once with a Bio-Rad Gene Pulser. The cells were again cooled on ice for 10 minutes, the cells were suspended in 20 ml of 10% FCS-MEM, and cultured in a 175 cm ² culture bottle at 37 ° C. in the presence of 5% CO ₂ . After 24 hours, freeze-thawing of this culture was repeated 3 times to recover the virus.

Recombinant viruses were selected from the recovered viruses by an erythrocyte adsorption test (HA test). The test method is as follows. The virus was inoculated into RK13 cells monolayer-cultured in a 9 cm Petri dish at 600 plaques / dish, and the cells were cultured for 2 days to form a plaque. The culture supernatant was removed and 0.5% chicken red blood cell solution was added. After incubating at 37 ° C. for 10 minutes, plaques were observed to obtain plaques (recombinant virus candidate strain) that did not adsorb erythrocytes.

Next, using the gene of the non-A non-B hepatitis virus structural protein region as a probe, 12 clones out of 34 clones of the recombinant virus candidate strain obtained above were subjected to plaque hybridization. Recombinant viruses with integrated genes were selected. First,
Recombinant virus candidate strains were inoculated at 20 to 50 plaques / dish on RK-13 cells monolayer-cultured in a 3 cm dish and cultured for 2 days to form plaques. A nylon membrane (Hybond N, manufactured by Amersham Co.) is placed on the formed plaque, the plaque is transferred onto the membrane, and treated with 0.5N NaOH for 5 minutes D
After denaturing NA, neutralize with 1M Tris-HCl (pH 7.4),
Further, it was treated with 1.5M NaCl and 0.5M Tris-HCl (pH 7.4) to adsorb the DNA to the membrane. After air-drying the membrane, it was irradiated with ultraviolet rays of 305 nm for 5 minutes to immobilize the DNA on the membrane. The membrane was immersed in hybridization buffer and incubated at 65 ° C for 1 hour. Furthermore, the membrane was transferred to a hybridization buffer containing a non-A non-B hepatitis B virus structural protein region gene labeled with ³² P using the random priming labeling method, and the mixture was reacted overnight at 65 ° C. to recombine with the probe DNA. Viral DNA was hybridized. The cells were washed twice with 1 × SSC, 0.1% SDS and 65 ° C. for 10 minutes each, and autoradiography was performed at −70 ° C. to detect positive clones.

As a result, 5 clones were positive, and each clone was designated as P7.5-E12 / RLV # 7, 8,
It was named 9, 10, 11.

Example 5 Confirmation of Expression by Indirect Fluorescent Antibody Method Monolayer-cultured RK-13 cells were treated with 0.05% trypsin.
After treatment with 1 mM EDTA solution to make single cells, ME
M culture solution (5% calf serum, 0.22% sodium bicarbonate)
Disperse to 50,000 cells / ml. Vaccinia parent strain Lister strain and P7.5-E12 / RL were added to this cell solution.
V is inoculated separately so that moi = 0.1. The cells inoculated with the virus are placed on a 12-well slide glass at 20 μl / well and cultured overnight at 37 ° C. under 5% CO ₂ . The cultured slide glass was washed once with distilled water, air-dried, and then -2.
Immerse in 5% acetone / 50% methanol mixed solution at 0 ° C. for 15 minutes to immobilize. After fixing, air dry and 50 with PBS (-)
20 μl / well of non-A, non-B hepatitis patient serum diluted 1-fold is added and reacted at 37 ° C. for 40 minutes. After 40 minutes of reaction, the plate was washed 3 times with PBS (-), and 250 times PBS (-) was added.
Anti-human IgG / FITC labeled (goat) diluted with 20
Add μl / well and incubate for 30 minutes at 37 ° C. After the reaction was completed, the cells were washed three times with PBS (-) and observed with a fluorescence microscope. No fluorescence was observed in cells infected with the Lister strain, but strong specificity was observed in cells infected with P7.5-E12 / RLV. Fluorescence was observed (Fig. 2).

Example 6 Confirmation of expression by immunoprecipitation method RK-13 cells were cultured overnight in 5% bovine serum EMEM medium to form monolayers. Lister stock or P
100 μCi was added to EMEM containing 7.5 mM E12 / RLV and 10 mM fructose instead of glucose.
³ H-glucosamine was added and the cells were cultured for 16 hours. SDS-polyacrylamide gel electrophoresis (SD) was performed by precipitating a specific antigen protein by immunoprecipitation using normal human serum or non-A non-B hepatitis patient serum and protein A sepharose.
S-PAGE). As a result, P7.5-E12
It was confirmed that the gp35-specific band was observed and expressed only in the cells immunoprecipitated from the / RLV-infected cells with the serum of non-A non-B hepatitis patients (FIG. 3).

Example 7 P7.5-E12 / RLV Immunization Immunization with rabbit serum
Sedimentation test P7.5-E12 / RLV was performed using Japanese white rabbit (1.5
(~ 2.0 kg) was intradermally inoculated with 10 ⁸ PFU and serum was collected 2 months later.

On the other hand, the Bluescript KS vector (St
C10-E12 DNA is inserted downstream of the T7 promoter of rategene) and C10-E12 DNA is inserted by in vitro transcription.
RNA complementary to E12 DNA was synthesized. This synthesized R
Using NA, in vitro translation was performed in a rabbit reticulocyte extract to synthesize a polypeptide encoded by C10-E12 DNA in vitro.

An immunoprecipitation test was conducted in the same manner as in Example 6 using the rabbit immune serum obtained above and the synthesized polypeptide (FIG. 4).

As a result, as shown in lane 2, P7.5
-E12 / RLV immunized rabbit serum contains C10-E12 DN which encodes the non-A non-B hepatitis virus structural protein region.
It was confirmed that an antibody specifically reacting with the polyprotein produced from A was produced.

[0057]

INDUSTRIAL APPLICABILITY The present invention enables the expression and production of non-A non-B hepatitis virus antigen proteins, particularly glycoproteins such as envelope proteins, and is therefore useful for treating or preventing non-A non-B hepatitis. , Has provided a vaccine antigen that can provide effective immunization.

[Brief description of drawings]

FIG. 1 shows a method for producing a recombinant plasmid and a recombinant vaccinia virus. In the figure, p22 is a gene encoding the core on the non-A non-B hepatitis virus genome,
gp35 is a gene encoding an envelope, and g
p75 represents a gene encoding a nonstructural protein.

FIG. 2 shows vaccinia virus Lister strain (control) or P7.5-E12 / RLV infected cells in non-A.
It is a photograph showing the result of fluorescent antibody staining using FITC-labeled anti-human IgG antibody (goat) after reacting with serum of non-hepatitis B patient.

FIG. 3 is a photograph showing the results of an immunoprecipitation test of vaccinia virus Lister strain (control) or P7.5-E12 / RLV infected cells with normal human serum or non-A non-B hepatitis patient serum. is there. However, lanes 1 and 3: Lister strain infected cells, lanes 2 and 4: P7.5-E12 / RLV infected cells, lanes 1 and 2: normal human serum, lanes 3 and 4: non-A non-B hepatitis patient serum. The numbers on the left end of the figure indicate molecular weight markers (using Amersham's Rainbow Marker).

FIG. 4 shows in vitro translation of P7.5-E12 / RLV immunized rabbit serum or normal human serum and C10-E12 DNA encoding the structural protein region of non-A non-B hepatitis virus. 3 is a photograph showing the result of immunoprecipitation test with the polyprotein produced by and the result of electrophoresis of the synthetic polyprotein. Lane 1
The description of 5 is as follows. Lane 1: In vitro translation is performed in the presence of a canin microsome membrane, sugar chain addition and processing of the synthesized protein are performed, and the results of an immunoprecipitation test between the obtained synthetic protein and normal human serum are shown. Since there is no antibody in normal human serum, no specific precipitation is observed. Lane 2: synthetic protein described above and p7.5-E12 / RL
The result of the immunoprecipitation test with V immunized rabbit serum is shown. 35k due to the presence of antibodies against gp35 in rabbit serum
A specific band is observed at the molecular weight of Da. Lane 3: shows the result of electrophoresis of the above synthetic protein.
A band indicating the presence of gp35 is observed at a position corresponding to the molecular weight of 35 kDa. Lane 4: shows the result of electrophoresis of a synthetic protein obtained by performing in vitro translation in the absence of a canin microsome membrane. Since the synthesized protein is not processed, p22 is added at both ends of gp35.
And a part of gp75 are added to the protein. Lane 5: RNA during in vitro translation
The result of the electrophoresis of the control which did not add is shown. No band appears because the protein is not synthesized. In addition, the numbers at the left end in the figure indicate molecular weight markers.

Continuation of front page (51) Int.Cl. ⁵ Identification number Office reference number FI Technical display location C12N 5/10 15/39 15/51 C12P 21/00 C 8214-4B // C12N 15/86 (C12P 21 / 00 C12R 1:91) (72) Inventor Michiho Ohara 1-3-1 Nishitsurugaoka, Oi-cho, Iruma-gun, Saitama Tonen Corporation Research Institute

Claims (18)

[Claims] 1. A gene containing a nucleotide sequence encoding a non-A non-B hepatitis virus antigen protein, a viral promoter capable of expressing the gene, and a vaccinia virus gene that is not essential for the growth of vaccinia virus. A plasmid that 2. The plasmid according to claim 1, wherein the antigen protein is a viral structural protein. 3. The plasmid according to claim 2, wherein the structural protein is an envelope glycoprotein. 4. The plasmid according to claim 1, wherein the viral promoter is a vaccinia virus-derived promoter. 5. The plasmid according to claim 1, wherein the vaccinia virus gene is a hemagglutinin gene. 6. The plasmid according to claim 1, wherein at least one set of a gene containing a nucleotide sequence encoding the antigen protein and the viral promoter is inserted into the vaccinia virus gene. 7. The plasmid P7.5-E12. 8. A recombinant vaccinia virus obtained by homologous recombination between the plasmid according to any one of claims 1 to 7 and vaccinia virus. 9. Recombinant vaccinia virus P7.5-E
12 / RLV. 10. A method for producing the recombinant vaccinia virus according to claim 8 or 9, wherein the plasmid according to any one of claims 1 to 7 is linearly restricted to cause infection with vaccinia virus. Transfecting a living animal cell for homologous recombination, and screening and recovering a recombinant virus in which a gene containing a nucleotide sequence encoding a non-A non-B hepatitis virus antigen protein is inserted Including the method. 11. The method according to claim 10, wherein the vaccinia virus is a vaccinia virus Lister strain. 12. The animal cell is a rabbit renal passage cell RK.
The method according to claim 10, which is -13. 13. An animal cell is infected with the recombinant vaccinia virus according to claim 8 or 9, and the cell is cultured to obtain non-A.
A method for producing an expressed non-A non-B hepatitis virus antigen protein, which comprises expressing a gene containing a nucleotide sequence encoding a non-B hepatitis virus antigen protein and recovering the expressed protein. 14. The animal cell is a rabbit kidney passage cell RK.
14. The method according to claim 13, which is -13. 15. An expressed non-A non-B hepatitis virus antigen protein having a sugar, which is obtained by the method according to claim 13. 16. An expression non-A having the sugar according to claim 15.
A vaccine for treating or preventing non-A non-B hepatitis, which comprises a non-B hepatitis virus antigen protein. 17. A vaccine for treating or preventing non-A non-B hepatitis, which comprises the attenuated recombinant vaccinia virus according to claim 8 or 9. 18. A vaccine for treating or preventing non-A non-B hepatitis, which comprises the inactivated recombinant vaccinia virus according to claim 8 or 9.