CN102304529A - Preparation method of rabbit hemorrhagic fever virus empty capsid antigen - Google Patents

Abstract

The invention relates to the field of genetic engineering, in particular to a preparation method of rabbit hemorrhagic fever virus empty capsid antigen. The method according to the present invention comprises the following steps: 1) constructing a baculovirus transfer vector comprising the capsid protein VP60 gene of rabbit hemorrhagic fever virus or an optimized gene, wherein codon optimization is carried out according to the codon frequency of silkworm; 2) constructing The obtained transfer expression vector is co-transfected with the baculovirus DNA to undergo homologous recombination or transposition to obtain the recombinant baculovirus; 3) Infect the insect host cell with the recombinant baculovirus; 4) Cultivate the infected insect host , expressing the corresponding empty capsid antigen of rabbit hemorrhagic fever virus, harvesting and purifying the expressed antigen. The method of the invention can greatly reduce the production cost of the rabbit hemorrhagic fever virus empty capsid antigen, and has many advantages such as safety, high efficiency, less energy consumption, and low cost.

Description

A kind of preparation method of empty capsid antigen of rabbit hemorrhagic fever virus

technical field

The invention relates to the field of genetic engineering, in particular to a preparation method of rabbit hemorrhagic fever virus empty capsid antigen.

Background technique

Rabbit viral hemorrhagic disease, commonly known as rabbit plague, is an acute, virulent, highly contagious and fatal infectious disease caused by rabbit hemorrhagic disease virus (RHDV). It was once a devastating infectious disease of rabbits. , has attracted the attention of the rabbit industry because of bringing huge economic losses to the rabbit industry. The disease was first reported in China in 1984. In 1989, the World Organization for Animal Health (OIE) officially listed the disease as a Class B infectious disease, and my country classified it as a Class II infectious disease (Wang Yongkun et al., Diagnosis and Prevention of Rabbit Distemper, 1992).

In the seventh report of the International Committee on Taxonomy of Viruses (ICTV) in 2000, rabbit hemorrhagic fever virus was included in the genus Lagovirus of the family Caliciviridae. RHDV virion is spherical, without envelope, 32-36nm in diameter, icosahedral symmetry (Zhang Lihong et al., Guangdong Animal Husbandry and Veterinary Science and Technology, 31:9-11, 2006), and its capsid consists of 32 cylindrical shells with a height of 5-6nm Granules consist of multiple copies of the major structural protein VP60. The surface of the negative-stained electron microscope has the typical cup-shaped structure of the calicivirus, and a small number of empty capsids without cores can also be seen under the electron microscope.

The RHDV genome contains two open reading frames, and the long open reading frame (ORF1) at the 5' end encodes a polyprotein precursor of 2344 amino acids, which is further decomposed into capsid protein and multiple non-structural proteins by viral proteases. The capsid protein is the main structural protein of the virus, called VP60, which is directly related to the induction of immune responses against viral infection. The short open reading frame (ORF2) at the 3' end, located between 7025-7378bp, encodes another small structural component of the virus, called VP10.

The N-terminal of VP60 constitutes the inner region of the capsid protein, and the C-terminal constitutes the outer part of the capsid protein. The N-terminal amino acid between 31-250 is the main anti-infection immunodetermining region. Studies on VP60 protein have found that when the capsid protein is expressed in vitro, it can naturally polymerize into virus-like particles that do not wrap nucleic acid and are physically similar to natural RHDV virions in the absence of any other components (Rob N, Virus-like particle asimmunogens, Trends in Microbiology, 11:438-444, 2003).

In recent years, the use of virus-like particles as antigenic carriers of vaccines has attracted more and more attention from scientists. Virus-like particles are chimeric particles that mimic the morphological characteristics of virus particles and present foreign gene fragment products on the surface of virus particles. , At the same time, it can also present specific antigens of other pathogens on its surface, so it plays an important role in the research of pathogen vaccines. VLPs can often induce a stronger humoral immune response than inactivated vaccines and soluble peptides, because the surface of VLPs presents glycoprotein antigens in a non-infectious particle state that mimics the natural antigen presentation process, and the presentation caused by this method The immune response is better than the dissolved state, so the immune response caused by the glycoprotein antigen, which plays an important role in protection, is expected to be closer to natural infection. In this way, virus-like particles are more likely to be widely used in the development of vaccines. Moreover, VLPs are not infectious because they do not encapsulate nucleic acid and cannot replicate, so they are a safe antigen carrier (Nagesha HS, Virus-like particles of calicivirus asepitope carriers, Arch Virol, 144: 2429-2439, 1999).

A large number of experiments have shown that RHDV cannot proliferate on chicken embryos, and it is difficult to proliferate stably in various primary or passage cells. The currently widely used vaccines are tissue-inactivated vaccines. Recently, research on novel vaccines has mainly focused on the development of genetically engineered vaccines, and VP60 proteins have been expressed in various expression systems such as Escherichia coli, Saccharomyces cerevisiae, vaccinia virus and plants (Liu Huairan et al. 9, 2003). Boga et al. expressed the main capsid protein VP60 of the RHDV Spanish AST/89 strain in E. coli and found that when VP60 was expressed as a β-galactosidase fusion protein, it only had part of the same antigen as the natural VP60 and did not induce protection Immunity; and in the expression system based on T7 RNA polymerase, it can express a protein that is very similar to the natural VP60 antigen and can induce effective protective immunity, which can resist the lethal attack of purified RHDV; Boga et al. Yeast expressed VP60 and could form virus-like particles; Famos et al. highly expressed VP60 of Spanish isolate AST/89 in yeast, the protein expression was as high as 1.5g/L and about 70% of the expressed protein was glycosylated; Yan Weiwei et al inserted the RHDV VP60 gene into pPICZ B and the recombinant protein expressed by Pichia pastoris has hemagglutination properties and can be inhibited by anti-RHDV hyperimmune serum (Yan Weiwei et al., Chinese Journal of Veterinary Medicine, 23:447-449, 2003). Fernandez-Femandez et al. successfully expressed VP60 using the vector constructed by Plum Pox Potato Y virus, and the expression product inoculated rabbits could resist the lethal dose of RHDV attack (Fernandez-Fernandez M R, Protection of rabbits against rabbit hemotthagic disease virus by immnization with the VP60 protein expressed in plants with a potyvirus based vector, Virology, 280: 283-291, 2001); Castanon et al. used potato-expressed VP60-containing extracts to immunize rabbits, which produced specific antibody responses and provided strong virulent attack against RHDV. Protection (Castanon S, The effect of the promoter on expression of VP60gene from rabbit hemorrhagic disease virus in potato plants, Plant Science, 162:87-95, 2002).

At present, there are still some problems in the research of RHDV genetically engineered vaccines: VP60 expressed by Escherichia coli has the disadvantages of insolubility and relatively poor immune effect, and its application prospect is not optimistic; Problem: The expression level of VP60 produced by transgenic plants is not ideal at present; therefore, it is imperative to develop a safe and efficient new vaccine for RHD.

Rabbit plague is characterized by respiratory system hemorrhage, solid organ edema, congestion and hemorrhagic changes. Infected rabbits often die within 48-72 hours. Preliminary diagnosis can be made according to the lesion characteristics, combined with laboratory detection of virus antigens in dead rabbit tissues for diagnosis, mainly using hemagglutination test (HA), enzyme-linked immunosorbent assay (ELISA), immunoelectron microscopy and molecular biology methods.

Since the establishment of the insect baculovirus expression vector system (BEVS) in 1983 (Smith, Mol. Cell Biol., 3: 2156-2165, 1983), nearly a thousand foreign genes have been expressed in the system. Its advantages are: A: BEVS has high expression efficiency, high biological activity of the expressed product, and its antigenicity and immunogenicity are similar to natural proteins; B: Bombyx mori baculovirus can accommodate large foreign genes without affecting itself C: Using late-late polyhedrin gene promoter to express exogenous gene, even if the recombinant gene product is toxic to cells, the expression level will not be affected; D: Co-infection with multiple expression vectors or several different recombinant viruses, It can express two or more foreign proteins at the same time, and study the supramolecular assembly of peptide chains and the structure and function of protein oligomers; E: Baculoviruses are not pathogenic to vertebrates, silkworms have no zoonotic diseases, It is considered as a genetically safe expression vector (Jing Zhizhong et al., China Veterinary Science and Technology, 31: 43-45, 2001).

The silkworm BmNPV expression system (Bombyx mori bioreactor) has been widely used in medicine, veterinary medicine and agriculture since it was developed in 1984. In 1997, Tian E et al. used the modified BmNPV as a vector to successfully express the 28KD glutathione S-transferase gene of Schistosoma japonicum mainland China strain in silkworm cells and larvae (Acta Biochemistry and Biophysics, 1997, 29 (1 ): 33-38). Zhang Chuanxi et al expressed the EPO gene in silkworm cultured cells and larvae, and the expressed product had good in vitro activity. In 1990, Chu Ruiyin and others expressed the HBsAg gene in silkworm cultured cells and their culture fluid respectively. Zhou Naiming et al. successfully expressed HBsAg in silkworm larvae and pupae, with an average yield of about 750 μg per silkworm and 690 μg per pupa, and the product mainly exists in the form of glycosylation. Dozens of animal virus antigenic proteins, such as foot-and-mouth disease virus, have been expressed and purified successively, and a considerable part of them is being commercialized (Li Zhiyong et al., Plos one, e2273, 2008). The prophylactic recombinant IL preparation (Intercat) for pets (cats and dogs) produced by the BmNPV system has been produced and put into the market by Japan "TORAY" Co., Ltd. 124-127).

Nowadays, the technology related to the silkworm baculovirus expression system is relatively mature. This system expresses foreign genes, which is not only economical and efficient, but also provides a new technical approach. The empty capsid antigen of rabbit hemorrhagic fever virus was produced by silkworm bioreactor to ensure its normal protein structure and biological immune activity, laying the foundation for the commercial production of rabbit hemorrhagic fever vaccine.

Contents of the invention

The inventors of the present invention proposed and completed the present invention in order to solve the above-mentioned problems.

The purpose of the present invention is to overcome the deficiencies of the prior art and provide a method for safely and efficiently expressing the empty capsid antigen of rabbit hemorrhagic fever virus in insects using the silkworm baculovirus expression system.

Another object of the present invention is to provide an optimized gene of capsid protein VP60 of rabbit hemorrhagic fever virus.

Another object of the present invention is to provide a baculovirus transfer vector and a recombinant baculovirus comprising capsid protein VP60 of rabbit hemorrhagic fever virus or the above-mentioned optimized gene.

Another object of the present invention is to provide an insect bioreactor for preparing rabbit hemorrhagic fever virus empty capsid antigen.

Another object of the present invention is to provide the rabbit hemorrhagic fever virus empty capsid antigen prepared by the above method.

Another object of the present invention is to provide the rabbit hemorrhagic fever virus empty capsid antigen prepared by the above method as a rabbit hemorrhagic fever vaccine.

According to the optimized gene of rabbit hemorrhagic fever virus capsid protein VP60 of the present invention, its nucleotide sequence is shown in SEQ ID No.2.

Further, the present invention provides a baculovirus transfer vector comprising capsid protein VP60 of rabbit hemorrhagic fever virus or the above-mentioned optimized gene.

The present invention also provides a recombinant baculovirus obtained by infecting a baculovirus with the above-mentioned baculovirus transfer vector.

The present invention also provides an insect bioreactor for preparing rabbit hemorrhagic fever virus empty capsid antigen, which is obtained by infecting an insect host with the above-mentioned recombinant baculovirus.

Therefore, the preparation method of rabbit hemorrhagic fever virus empty capsid antigen according to the present invention comprises the following steps:

1) Constructing a baculovirus transfer vector comprising the capsid protein VP60 gene of rabbit hemorrhagic fever virus or the above-mentioned optimized gene, wherein the codon is optimized according to the codon frequency of silkworm;

2) co-transfecting the constructed transfer expression vector with baculovirus DNA to obtain homologous recombination or transposition to obtain recombinant baculovirus;

3) Infecting insect host cells with the recombinant baculovirus;

4) culturing the infected insect host, expressing the corresponding rabbit hemorrhagic fever virus empty capsid antigen, harvesting and purifying the expressed antigen.

According to the preparation method of the rabbit hemorrhagic fever virus empty capsid antigen of the present invention, wherein, the original sequence of the rabbit hemorrhagic fever virus capsid protein VP60 gene and the nucleotide sequence after codon optimization are as SEQ ID NO.1 And shown in SEQ ID NO.2, the aminoacid sequence SEQ ID NO.3 of the viral VP60 of its encoding.

The preparation method of rabbit hemorrhagic fever virus empty capsid antigen according to the present invention comprises, wherein, described baculovirus transfer carrier is preferably selected from AcRP23-lacZ, AcRP6-SC, AcUW1-lacZ, BacPAK6, Bac to Pac, Bacmid, p2Bac , p2Blue, BlucBacH (pETL), p89B310, pAc360, pAc373, pAcAB3, pAcAB4, PAcAS3, pAcC129, pAcC4, DZI, pAcGP67, pAcIE1, pAcJP1, pAcMLF2, pAcMLF7, pAcMLF8, pAcMP1, pAcMP2, pAcRP23, pAcRW45, pAcsMAG、pAcUW1、pAcUW21、pAcUW2A、pAcUW2B、pAcUW3、pAcUW31、pAcUW41、pAcUW42、pAcUW43、pAcUW51、pAcVC2、pAcVC3、pAcYM1、pAcJcC5、pBac1、pBac2、pBlueBacIII、pBlueBacHis、pEV55、mXIV、pIEINeo、pJVETL、pJVNhe1、pJVP10、 or Or other similar baculovirus homologous recombination or transposable vectors, more preferably pVL1393 baculovirus carrier vector.

According to the preparation method of rabbit hemorrhagic fever virus empty capsid antigen of the present invention, wherein, the constructed transfer vector is the vector pVL1393-RHDV-VP60 with the original sequence of rabbit hemorrhagic fever virus capsid protein VP60 and the carrier pVL1393-RHDV-VP60 with rabbit hemorrhagic fever virus The vector pVL 1393-RHDV-VP60-O of the codon-optimized sequence of the capsid protein VP60 gene.

According to the preparation method of rabbit hemorrhagic fever virus empty capsid antigen of the present invention, wherein, described baculovirus is preferably selected from BmNPV, AcMNPV, ApNPV,, HaNPV, HzNPV, LdMNPV, MbMNPV, OpMNPV, SlMNPV, SeMNPV or SpltNPV, more Preferably it is the parent strain of Bombyx mori baculovirus Bm-NPV-ZJ8.

According to a preferred embodiment of the present invention, wherein, the recombinant baculovirus is selected from any of the following recombinant viruses: (1) recombinant Bombyx mori nuclear polyhedrosis virus rBmNPV (RHDV-VP60), (2) recombinant Bombyx mori karyotype Polyhedrosis virus rBmNPV (RHDV-VP60-O).

According to the preparation method of rabbit hemorrhagic fever virus empty capsid antigen of the present invention, wherein, described insect host is selected from Lepidoptera insects, such as silkworm (Bombyx mori), wild silkworm (Bombyx mandarina), castor silkworm (Philosamiacynthia ricim) , Dictyoplca japanica, Philosamia cynthia pryeri, Antheraea pernyi, Antheraea yamamai, Antheraea polyphymus, Atographa califorica, Ectropis obliqua, Cabbage moth (Mamestrabrassicae), Spodoptera littoralis, Fall Armyworm (Spodoptera frugiperda), Trichoplusia ni, Armyworm (Thaumetopoea wilkinsoni), Cotton bollworm (Heliothis armigera), American bollworm (Heliothis zea), tobacco budworm (Heliothis assulta), tobacco moth (Heliothis virescens), oriental armyworm (Pseudaletia separata), gypsy moth (Lymantria dispar), etc.; more preferably silkworm (Bombyxmori).

According to the preparation method of rabbit hemorrhagic fever virus empty capsid antigen of the present invention, wherein, described infection refers to that recombinant baculovirus infects 1-5 age insect larvae or pupae (more preferably : Infect silkworm cells with recombinant silkworm baculovirus or puncture inoculate silkworm larvae or pupae of 1-5 instars, collect the body fluid or tissue homogenate of silkworm larvae or pupae containing rabbit hemorrhagic fever virus empty capsid antigen after 3-6 days of infection ); Wherein, described pupal body optimum is the early tender pupa of 1-2 days.

The present invention adopts gene recombination technology to optimize the codons of the capsid protein gene sequence of rabbit hemorrhagic fever virus (RHDV), the original sequence (SEQ ID NO: 1) and the optimized sequence (SEQ ID NO: 2) of the protein ) into a baculovirus transfer vector (such as AcRP23-lacZ, AcRP6-SC, AcUW1-lacZ, BacPAK6, Bac to Pac, Bacmid, BlueBacII (pETL), p2Bac, p2Blue, p89B310, pAc360, 373, pAcAB3, 4, pAcAS3 , pAcC129, C4, DZ1, pAcGP67, pAcIE1, pAcJP1, pAcMLF2, 7, 8, pAcMP1, 2, pAcRP23, 25, pAcRW4, pAcsMAG, pAcUW1, 21, 2A, 2B, 3, 31, 41, 42, 43, 51 , pAcVC2, 3, pAcYM1, pAcJcC5, pBac1, 2, pBlueBacIII, pBlueBacHis, pEV55, mXIV, pIEINeo, pJVETL, pJVNhe1, pJVP10, pJVrsMAG, pMBac, pP10, pPAK1, pPBac, pSHONEX 1.1, pSYN XIV VI+, pSYNVI+ pSYNXIV VI-, pVL1391, 1392, 1393, pVL941, 945, 985, pVTBac, pBM030, pUAC-5), under the control of the polyhedron promoter, p10 promoter or other strong promoters from viruses and eukaryotes , through in vivo/in vitro recombination, the original sequence and optimized sequence of the RHDV capsid protein are integrated into the genome of the baculovirus to obtain a recombinant virus; the recombinant virus can be obtained by adding food or using various Means to infect 1-5 instar insect larvae or pupae (optimum time is 1-2 days early tender pupa) through the epidermis to express and produce empty capsid antigen of rabbit hemorrhagic fever virus .

According to a preferred embodiment of the present invention, the capsid protein gene sequence of rabbit hemorrhagic fever virus (RHDV) is codon-optimized, the original sequence of the capsid protein and the optimized sequence, i.e. SEQ ID NO.1 and SEQ ID NO The base sequence shown in .2 was cloned into the baculovirus carrier pVL1393, and then the original sequence VP60 of the capsid protein gene of rabbit hemorrhagic fever virus and the codon-optimized sequence VP60-O were transferred to the silkworm rod through in vivo recombination. On the genome of the parental virus strain Bm-NPV-ZJ8, the Polyhedrin gene on the genome was replaced, and the recombinant Bombyx mori baculovirus rBmNPV (HDV- VP60), rBmNPV (HDV-VP60-O); infect silkworm cell lines or puncture inoculate 1-5 instar silkworm larvae or pupae, and multiply rBmNPV (HDV-VP60), rBmNPV (RHDV-VP60-O); when When rBmNPV (RHDV-VP60) and rBmNPV (RHDV-VP60-O) replicate in silkworms, the VP60 and VP60-O genes are expressed under the control of the polyhedrin gene (polh) promoter, and self-assemble into rabbit haemorrhagic fever virus empty Capsid antigen; after 3-6 days of infection (optimally 5 days, 25°C feeding temperature), the body fluid (or whole homogenate) of silkworm larvae or pupae containing rabbit haemorrhagic fever virus empty capsid antigen is collected and irradiated After the baculovirus is killed and the protein is purified, a safe and efficient rabbit hemorrhagic fever virus empty capsid antigen can be obtained, and the antigen can be used to prepare an injection vaccine for preventing rabbit viral hemorrhagic disease.

The method of the present invention adopts the baculovirus expression system to safely and efficiently produce the rabbit hemorrhagic fever virus antigen in the silkworm bioreactor, and its production cost is significantly lower than the traditional method for preparing the rabbit hemorrhagic fever virus antigen, and the construction of a factory requires three wastes, electricity and Energy consumption such as water resources is minimal. Since the silkworm has been approved by the Ministry of Health of my country as an insect that can be used both as food and medicine, the antigen prepared by the method of the present invention is highly safe after being purified, and can be directly prepared as a vaccine for immunizing animals.

Generally speaking, the method of the present invention can greatly reduce the production cost of rabbit hemorrhagic fever virus empty capsid antigen, and has many advantages such as safety, high efficiency, less energy consumption, and low cost.

Description of drawings

Fig. 1 is the scanning electron micrograph of the RHDV empty capsid particle that silkworm produces;

Fig. 2 is an electron microscope scanning image of an empty capsid produced by silkworm by immunogold labeling.

Detailed ways

The present invention is produced in detail below in conjunction with the embodiments and accompanying drawings, and those skilled in the art can understand that these examples are entirely illustrative and do not limit the protection scope of the present invention.

Experimental Materials

Escherichia coli strain E.coli DH _5α was purchased from Promega Company; cloning vector pEASY-T3 was purchased from Quanshijin Company; cloning vector pMD18-T was purchased from TaKaRa Company; prokaryotic expression vector pET-28a ⁺ , recipient strain E.coli TOP10 and BL21(DE3) are both conventional strains purchased from Novagen, and the carrier pVL1393 was purchased from Invitrogen; the original sequence VP60 of the capsid protein gene of rabbit hemorrhagic fever virus was extracted from diseased animal lesion tissues and cloned onto the carrier The codon-optimized sequence VP60-O was obtained by direct gene synthesis; the silkworm cell BmN and the silkworm nuclear polyhedrosis virus parent strain Bm-NPV-ZJ8 were donated by researcher Wu Xiangfu, Academy of Life Sciences, Chinese Academy of Sciences, Molecular Biotechnology Institute, Chinese Academy of Agricultural Sciences Preserved in the microbiological laboratory; the highly expressed silkworm variety JY1 was bred by the Sericulture Science Research Institute of the Chinese Academy of Agricultural Sciences and preserved by the Institute of Biotechnology of the Chinese Academy of Agricultural Sciences.

Enzymes and reagents: the restriction endonucleases and buffers used were purchased from Promega; T4 DNALigase and buffers were purchased from Promega; LA Taq polymerase and buffers were purchased from TaKaRa; RNaseA and dNTPs were purchased from Sigma ; DNA and protein molecular weight standards of various specifications are products of TranGenBiotech Company; DEPC, M-MLV-Rtase (reverse transcriptase) are purchased from Promega Company.

Biochemical reagents: bisacrylamide, acrylamide, IPTG, X-Gal were purchased from Promega; Tris, Ampicillin, Kanamycin, IPTG, SDS, urea, imidazole, TritonX-100, TEMED (N, N, N', N'-Tetramethylethylene diamine) , Ammonium Persulfate (Ammonium Persulfate), Kanamycin (Kanamycin), and cell culture medium TC-100 were purchased from Sigma Company; agarose was a product of Sunbiotech Company; OXOID company; 0.2um, 0.45um filter was purchased from Gelman Sciences company; Ethidium bromide (EB), Coomassie brilliant blue R-250 was purchased from Fluka company; Agar powder was imported from Japan; Ni-NTA resin, Proteinase K, Fetal bovine serum was purchased from Invitrogen; others were domestic or imported analytical reagents. Primers were synthesized by Sanbo Polygala Biotechnology Co., Ltd.

Culture medium: Escherichia coli medium is LB medium; silkworm cell culture medium is TC-100 medium.

The animal experiment of the capsid protein gene expression product of rabbit hemorrhagic fever virus was carried out in an isolated laboratory.

Example 1, Soluble expression of the original sequence VP60 of rabbit hemorrhagic fever virus capsid protein gene in silkworm bioreactor and detection of expression products

1. Acquisition of the original sequence VP60 of the capsid protein gene of rabbit hemorrhagic fever virus

TRIzol method was used to extract genomic RNA from rabbit diseased tissues.

Primers were designed to amplify the original sequence VP60 (SEQ ID NO.1) of the capsid protein gene of rabbit hemorrhagic fever virus by RT-PCR. The designed reverse transcription specific primer is: RHDV-RT: 5'-GGATTAAAACCTAACCTACC-3'. The amplification primers of the original sequence VP60 of the capsid protein gene are: VP60 upstream: 5'-G GAATTC AACATGGAGGGCAAAGCCCGCACAG-3' (EcoR I); VP60 downstream: 5'-GA AGATCT TCAGACATAAGAAAGCCATTG-3' (Bgl II).

After PCR amplification, the fragment size was analyzed by agarose gel electrophoresis. The target gene fragment was recovered and connected with the cloning vector (pMD18-T). Transform E.coli heat-shock competent cells and identify positive recombinant plasmids. Add the strain identified as a positive clone (named pT-RHDV-VP60) to the LB culture medium containing Amp again, and after shaking at 220r/min overnight, draw 1mL of fresh bacterial liquid into a sterile Eppendorf tube, add a small amount of Glycerol, after sealing with a parafilm, went to the sequencing department of Beijing Sanbo Biotechnology Co., Ltd. for sequencing. The result is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.3. The sequencing results were analyzed by DNAStar and DNAMAN software.

2. Construction of baculovirus transfer vector pVL1393-RHDV-VP60

The recombinant plasmid pT-RHDV-VP60 identified by sequencing was digested with EcoRI to linearize it. The eukaryotic expression vector plasmid pVL1393 was subjected to the same digestion treatment. Ligation of the RHDV-VP60 target fragment recovered by enzyme digestion with the transfer vector pVL1393 after EcoR I/Bgl II double enzyme digestion. The recombinant plasmid was digested with EcoR I/Bgl II, identified by bacterial liquid PCR and gene sequencing, and the correctly identified recombinant plasmid was named pVL1393-RHDV-VP60.

3. Propagation of Bombyx mori nuclear polyhedrosis virus parent strain Bm-NPV-ZJ8 and preparation of virus DNA

The culture medium was prepared according to the instructions of Sigma company, and the silkworm cell BmN was cultured at 27°C. Infect about 50ml of cells in the logarithmic growth phase with the parental strain of silkworm nuclear polyhedrosis virus Bm-NPV-ZJ8, and the multiplicity of infection is 1. After 3 to 4 days, collect the virus infection solution, centrifuge (10000rpm × 10min), remove the precipitate, and supernatant Centrifuge at 25000rpm for 1 hour, remove the supernatant, suspend the virus particle precipitation with 1ml virus DNA extraction solution (1000ml contains 12.1g Tris, 33.6g EDTA, 14.1g KCl, pH7.5), extract DNA, and store at 4°C spare.

4. Construction and acquisition of recombinant Bombyx mori baculovirus rBmNPV (RHDV-VP60)

Inoculate approximately 1× ¹⁰⁶ cells in a 15cm2 culture flask. After the cells adhere to the wall, remove the medium containing fetal bovine serum (FBS), wash three times with medium without FBS, and add 1.5ml of medium without FBS. Add 1 μg of silkworm baculovirus parent strain Bm-NPV-ZJ8 DNA, 2 μg of recombinant transfer plasmid pVL1393-RHDV-VP60DNA and 5 μl of liposomes to a sterilized tube in sequence, make up the volume to 60 μl with sterile double distilled water, and mix gently After standing for 15 minutes, it was added dropwise into the culture flask for co-transfection. Microscopic observation, the plaques without polyhedrons were selected, and the above steps were repeated to obtain pure recombinant Bombyx mori baculovirus rBmNPV (RHDV-VP60) through 2 to 3 rounds of purification.

The recombinant Bombyx mori baculovirus rBmNPV (RHDV-VP60) was used to infect normal growing BmN cells, and the supernatant was collected after 3 days of culture, which contained a large amount of recombinant virus rBmNPV (RHDV-VP60).

Use the PCR method to analyze the integration of foreign genes, and the oligonucleotide primers are:

VP60 upstream: 5'G GAATTC AACATGGAGGGCAAAGCCCGCACAG-3' (EcoR I); VP60 downstream: 5'-GA AGATCT TCAGACATAAGAAAGCCATTG-3' (Bgl II).

As a result, it was confirmed that recombinant virus was obtained.

6. High expression of RHDV-VP60 in silkworm chrysalis and silkworm body

The silkworm chrysalis used was JY1, a high-expression variety. The silkworm of JY1 variety was reared according to the conventional method of "Chinese Sericulture Science" (Shanghai Science and Technology Press, 1991) edited by Lu Hongsheng. 48 hours after feeding, silkworms with the same average body weight and 15 silkworm chrysalis with the same average weight after cocooning were selected, and each silkworm chrysalis and silkworm were inoculated with about 1.0×10 ⁵ rBmNPV (RHDV-VP60). Silkworm blood was frozen at -20°C for double-antibody sandwich ELISA detection.

7. Collection and purification of RHDV-VP60 virus-like particles.

The silkworm chrysalis expressing RHDV-VP60 was ground in a homogenizer with pre-cooled PBS (the ratio of solid to liquid was 1:9), and then filtered with a 0.45um filter. In 30% sucrose solution, ultracentrifuge at 1.5×10 ⁵ g for 2 hours. Redissolve the precipitate to the original volume with a solution of Tris-HCl (PH7.0) containing 0.1M NaCl, and elute with cation exchange chromatography packing material SP (GE Company), and Tris-HCl (PH7.0) of 0.5M NaCl . And then through molecular sieve chromatography S200 (GE Company). The purity can reach 95%, and the yield can reach more than 40%. At the same time, it is proved that the target protein expressed in silkworm can self-assemble into empty capsid particles of virus at high concentration, and a corresponding purification method of empty capsid antigen of rabbit hemorrhagic disease virus expressed in silkworm is established.

8. Detection of expression products

(1) ELISA and Western blotting detection

The silkworm blood was collected when the virus was injected into the silkworm. ELISA method was used to detect the expression level of RHDV-VP60 antigen protein, the coating liquid was used as blank control, normal silkworm blood was used as negative control, the rabbit serum after prokaryotic expression of RHDV-FR2 protein immunized New Zealand white rabbit was used as primary antibody, and HRP was labeled The goat anti-rabbit antibody was used as the secondary antibody. Randomly take samples from the collected silkworm blood, and use the coating solution for gradient dilution, from 50× to 6400 times, do double-hole detection at each gradient, take the average value when calculating, and take 100 μL for each coating onto the microtiter plate. The results are shown in Table 1, indicating that the RHDV-VP60 gene is highly expressed in silkworms, with an average expression level of at least 1 mg per silkworm or silkworm chrysalis, and the specificity of antigens and antibodies can still be detected at 3200-fold dilutions or even higher dilutions reaction.

Table 1 ELISA detection of VP-60 gene expressed in silkworm bioreactor

The results of Western blotting showed that a specific band with a size of 60kD could be detected in the supernatant of silkworm hemolymph samples after recombinant virus infection.

(2) Detection of hemagglutination assay of empty capsid antigen of rabbit hemorrhagic disease virus expressed in silkworm

Take human "O" type red blood cells and store them in Alfred's solution, wash the red blood cells three times with 20 times normal saline, and finally make the red blood cells into a 1% suspension with normal saline. Add 50ul of sterilized normal saline to each well of the U-shaped plate; use a micropipette to take 50ul of the dilution of the virus-containing empty capsid solution diluted by an appropriate multiple and add it to the first well, mix well, absorb 50ul and add it to the second well In the second well, serially dilute to the 10th well, absorb 50ul of liquid from the 10th well and discard; the 11th and 12th wells are normal saline control, and the negative silkworm blood is diluted in the same way. Add 50 ul of 1% human "O" red blood cell suspension to each well, mix well, react at 4°C for 45 minutes, and observe the result after the red blood cells in the control are completely deposited. When the preliminarily purified empty capsid antigen of rabbit hemorrhagic disease virus was diluted to 64000 times, it was still positive.

(3) Animal immunization experiments.

The collected purified antigen RHDV virus capsid particles were immunized by intramuscular injection and oral route to 10 rabbits respectively as the test group, and another 5 rabbits were only injected with adjuvant and fed with antigen as the control group, and the rabbits were all negative for RHDV . 5ug RHDV empty capsid antigen and oil adjuvant were mixed in equal volumes to make a trial vaccine, 1ml/only for animal experiments on rabbits, and blood was collected after 21 days for ELISA antibody titer testing. All the test groups produced antibodies against RHDV, and the injection group The detection titer can reach more than 3200 times; the initially purified empty capsid virions are mixed with the same volume of propolis containing 25%-35% fatty acid and 8%-12% according to the amount of 50 μg per rabbit, the best Concentration is fatty acid 30% + propolis 10% (fatty acid is composed of palmitic acid 8%, oleic acid 19%, other components are 3%), perfusion after ultrasonic emulsification, one month later, the IgA titer of the oral group also reached 1600 times . The results showed that 100% of the test group produced protective antibodies, but no antibodies were detected in the control group; the challenge experiment was carried out at the same time as blood collection, and no rabbits in the immune group died no matter whether they were injected or orally administered, while all rabbits in the control group died within 3 days after challenge. Typical symptoms of rabbit hemorrhagic fever.

Example 2, Soluble expression of capsid protein gene of rabbit hemorrhagic fever virus codon-optimized sequence VP60-O in silkworm bioreactor and detection of expression products

1. Obtaining the optimized sequence VP60-O

According to the silkworm codon preference, the original sequence VP60 of the rabbit hemorrhagic fever virus capsid protein gene measured in Example 1 is optimized without changing the amino acid sequence. After optimization, the sequence VP60-O is shown in SEQ ID NO.2, and in the original sequence Contains rare codons in tandem, which reduces the translation sequence or even untranslates the device. After optimization, the CAI value of the sequence is increased from 0.72 to 0.79. The GC content and unsuitable peaks are adjusted to prolong the half-life of mRNA. The GC content is adjusted from 54.9% to 53.34 %, those stem-loop structures that affect mRNA stability and its combination with ribosomes are destroyed, there are Bam HI restriction sites at position 90 in the original sequence, and Xho I restriction sites at position 487, the optimized sequence There are no such two enzyme cutting sites. The optimized sequence VP60-O was directly synthesized and cloned into the vector pUC57, with Bam HI and EcoR I restriction sites at both ends. After sequencing and identification, SEQ ID NO: 2 and the corresponding amino acid sequence SEQ ID NO.3 were obtained by analysis.

2. Construction of recombinant baculovirus transfer vector pVL1393-RHDV-VP60-O

The recombinant plasmid pUC57-RHDV-VP60-O identified by sequencing was digested with Bam HI to make it linearized. The eukaryotic expression vector plasmid pVL1393 was treated in the same way.

The plasmid pUC57-RHDV-VP60-O was ligated with the transfer vector pVL1393 after Bam HI/EcoR I double digestion and the transfer vector pVL1393 after Bam HI/EcoR I double digestion.

The ligation product was transformed into competent cells, and the correct recombinant plasmid was identified as pVL1393-RHDV-VP60-O.

3. Propagation of Bombyx mori nuclear polyhedrosis virus parent strain Bm-NPV-ZJ8 and preparation of virus DNA

With embodiment 1.

4. Construction and acquisition of recombinant Bombyx mori baculovirus rBmNPV (RHDV-VP60-O)

With embodiment 1.

The recombinant Bombyx mori baculovirus rBmNPV (RHDV-VP60-O) was used to infect normal growing BmN cells, and the supernatant was collected after 3 days of culture, which contained a large amount of recombinant virus rBmNPV (RHDV-VP60-O). The foreign gene integration was analyzed by PCR method. The oligonucleotide primers are: according to the optimized VP60-O gene sequence, two primers are designed to identify the sequence:

RHDV-F: 5'-ACCTTGGTCCTTTCCGTATATAA-3';

RHDV-R: 5'-CTTGGCGACAGTTTGAGCAC-3'.

As a result, it was confirmed that recombinant virus was obtained.

5. High expression of RHDV-VP60-O in silkworm chrysalis and silkworm body

The silkworm chrysalis used was JY1, a high-expression variety. The silkworm of JY1 variety was reared according to the conventional method of "Chinese Sericulture Science" (Shanghai Science and Technology Press, 1991) edited by Lu Hongsheng. 48 hours after feeding, select silkworms with the same average weight and 15 silkworm chrysalis with the same average weight seven days after cocooning. Each silkworm chrysalis and silkworm are inoculated with about 1.0×10 ⁵ rBmNPV (RHDV-VP60-O), and the infected silkworm chrysalis are collected 4-5 days later. And the silkworm blood was taken and stored at -20°C for double antibody sandwich ELISA detection.

6. Collection and purification of RHDV-VP60-O virus-like particles

The silkworm chrysalis expressing RHDV-VP60-O were ground in a homogenizer with pre-cooled PBS (the ratio of solid to liquid was 1:9), and then filtered with a 0.45um filter. In 30% sucrose solution, ultracentrifuge at 1.5×10 ⁵ g for 2 hours. The precipitate was redissolved to the original volume with a solution of Tris-HCl (PH7.0) containing 0.1M NaCl, and eluted through cation exchange chromatography packing material SP (GE Company) and Tris-HCl (PH7.0) of 0.5M NaCl. And then through molecular sieve chromatography S200 (GE Company). The purity can reach 95%, and the yield can reach more than 40%. At the same time, it is proved that the target protein expressed in the silkworm is soluble, and a corresponding purification method for expressing the empty capsid antigen of the genetically engineered rabbit hemorrhagic disease virus in the silkworm is also established.

7. Preparation of rabbit hemorrhagic disease virus detection antibody

Three pairs of specific primers were designed to express the optimized capsid protein sequence VP60-O in three segments for prokaryotic expression. The primers were designed as follows:

RHDV-F1: 5′-CGGGATCCATGGAGGGAAAAGCGAGGACAG-3′ (Bam HI)

RHDV-R1: 5′-CAAGCTTCTAGACCTGAATAGCATTGGTAGAAC-3′ (Hind III)

RHDV-F2: 5′-CGGGATCCACCTTGGTCCTTTCCGTATATAA-3′ (Bam HI)

RHDV-R2: 5′-CAAGCTTCTACTTGGCGACAGTTTGAGCAC-3′ (Hind III)

RHDV-F3: 5′-CGGGATCCACCGGAGCGCCCAACAATTTG-3′ (Bam HI)

RHDV-R3: 5′-CAAGCTTCTACACATACGAGAAACCGTTG-3′ (Hind III)

(1) Construction of recombinant plasmids pT3-RHDV-FR1, pT3-RHDV-FR2 and pT3-RHDV-FR3

Using the plasmid pUC57-RHDV-VP60-O as a template, use the above three pairs of primers to amplify the target fragments RHDV-FR1, RHDV-FR2, and RHDV-FR3; T3 connection, the connection product was transformed into the prepared Escherichia coli heat-shock competent cells, and the identification of the recombinant plasmid was carried out by spot-picking culture. After the rapid identification of the cell disruption method, the strain with the plasmid band was picked, and the recombinant plasmid pT3-RHDV was extracted by alkaline lysis. -FR1, pT3-RHDV-FR2 and pT3-RHDV-FR3 were identified by double enzyme digestion with Bam HI/Hind III, and the recombinant bacterial cultures with correct enzyme digestion identification were sent to Sanbo Yuanzhi Company for DNA sequencing verification. The sequencing results were compared with the sequence of the previous plasmid pUC57-RHDV-VP60-O, and the results showed that the target sequences in the three recombinant plasmids were consistent with the results of the previous plasmid sequence, and there were no frameshift mutations and the designed enzyme cutting site point.

(2) Construction of recombinant plasmids pET28a-RHDV-FR1, pET28a-RHDV-FR2, pET28a-RHDV-FR3

The correctly sequenced recombinant plasmids pT3-RHDV-FR1, pT3-RHDV-FR2 and pT3-RHDV-FR3 were digested with Bam HI/Hind III, purified and recovered by the glass milk method, and connected to the On the expression vector pET-28a digested by /Hind III double enzyme digestion, the ligation product was transformed into Top10 competent cells, and after spot picking, the cell disruption method was used for rapid identification, and then a small amount of alkaline method was used to quickly extract recombinant plasmids pET28a-RHDV-FR1, pET28a -RHDV-FR2, pET28a-RHDV-FR3, identified by double digestion with Bam HI/Hind III.

(3) Fusion expression induced by recombinant plasmid in Escherichia coli

The above-mentioned recombinant plasmids with correct digestion were transformed into BL21 competent cells respectively, and the bacterial liquid was collected after IPTG induction for 4 hours, and the expression was analyzed by SDS-PAGE electrophoresis. The results showed that compared with the negative control, the strains transformed with the recombinant plasmid pT3-RHDV-FR2 appeared The expression band of the fusion protein is the most concentrated. Pick the single colony of the expression strain with the best effect and induce it after shaking culture. After ultrasonic disruption, SDS-PAGE electrophoresis analysis shows that the expressed fusion protein (His-RHDV-FR2) exists in the precipitate, indicating that the expressed protein is in the form of inclusion bodies exist.

(4) Purification of fusion protein and preparation of its antibody

Pick a single colony of the successfully recombined expression strain and shake it to culture it to induce expression in large quantities. After the inclusion body is dissolved by using the relevant solution and method for processing the inclusion body protein, the expressed protein is purified by Ni+-NTA resin chromatography column. In urea NTA- 25. Urea NTA-50, urea NTA-100, urea NTA-250, urea NTA-500, 5 gradients to collect eluate, collect breakthrough liquid, eluate, collect one volume of NTA in each tube, analyze by SDS-PAGE Determine protein binding, distribution of target protein in eluate. The results showed a single band with the correct size. The size of the expressed target protein His-RHDV-FR2 was consistent with that of the positive control. When the urea NTA-100 was used for elution, the elution peak was the largest. SDS-PAGE purified the protein with a sterilized knife Cut off the purified protein band and cut it into small gel pieces of 1 mm ³ , and send it to the State Key Laboratory of Biofilm and Membrane Bioengineering, Institute of Zoology, Chinese Academy of Sciences for mass spectrometry analysis. The method is the database search and identification method of secondary mass spectrometry, and the results show The sample protein sequence detection coverage rate is 34.82%. The analysis shows that the Score Delta Cn of the sample exceeds 30, and there are 2 peptides that can be matched, and the amino acid sequence of 1 peptide is greater than 50. The AA sequence that can be detected matches the expected sequence The rate is as high as 34.68%, so it can be confirmed that the purified protein is the expression product of the target gene RHDV-FR2. The expressed fusion protein was purified and concentrated, and the concentration of the concentrated protein solution was measured by Bradford method. SDS-PAGE the purified fusion protein with a concentration of more than 1mg/ml and a total amount of not less than 3mg, cut off the target band, grind the micelle, and send it to the Institute of Genetics and Development, Chinese Academy of Sciences to immunize RHDV-negative rabbits Preparation of polyclonal antibodies.

(5) Polyclonal antibody titer detection

Purified RHDV-FR2 protein was immunized with New Zealand white rabbits for 4 times to obtain polyclonal antibody. The purified fusion protein was used as the coating antigen, and diluted with PBS in different times of 100, 200, 400, 800, 1600, 3200, 6400, 12800, 25600 , 51200 polyclonal antiserum as primary antibody, sandwich ELISA detection method to measure antibody titer diagram, rabbit serum before immunization as negative control, absorbance value at A492nm, take each dilution factor as the X axis, absorbance value under each dilution factor The mean is on the y-axis. Usually greater than 2.1 times the OD value of the specified negative control, it is positive (calculated after zeroing the blank control well). The results show that the titer of the self-made antibody can reach 1:3200 when the antigen protein concentration is 10 μg/ml .

8. Detection of expression products

(1) ELISA and Western blotting detection

The silkworm blood was collected when the virus was injected into the silkworm. ELISA method was used to detect the level of RHDV-VP60-O antigen protein expression. The coating solution was used as a blank control, and normal silkworm blood was used as a negative control. The rabbit serum after prokaryotically expressed RHDV-FR2 protein was immunized with New Zealand white rabbits was used as the primary antibody. HRP-labeled goat anti-rabbit antibody was used as the secondary antibody. Samples of the collected silkworm blood were randomly taken, and were serially diluted with the coating solution, from 100× to 6400 times. Double-hole detection was performed at each gradient, and 100 μL was taken to coat the microplate. The results are shown in Table 2, showing that the expression level of RHDV-VP60-O gene in silkworm is high, which is 2-3 times higher than that of RHDV-VP60 gene in silkworm, and can still be detected at 6400 times dilution or even higher dilution Antigen and antibody specific reaction (as shown in Table 2).

Table 2ELISA detects the VP60-O gene expressed in the silkworm bioreactor

The results of Western blotting showed that a specific band with a size of 60kD could be detected in the supernatant of the silkworm hemolymph sample after recombinant virus infection.

(2) Detection of hemagglutination assay of empty capsid antigen of rabbit hemorrhagic disease virus expressed in silkworm

Take human "O" type red blood cells and store them in Alfred's solution, wash the red blood cells three times with 20 times normal saline, and finally make the red blood cells into a 1% suspension with normal saline. Add 50ul of sterilized physiological saline to each well of the U-shaped plate; use a micropipette to take 50ul of the purified empty capsid antigen dilution solution and add it to the first well, mix well, absorb 50ul and add it to the second well, In this way, serially dilute to the 10th well, absorb 50ul of the liquid from the 10th well and discard it; the 11th and 12th wells are normal saline control, and the negative silkworm blood is diluted in the same way. Add 50ul of 1% human "O" red blood cell suspension to each well, mix well, and react at 4°C for 45 minutes, and observe the results after the control red blood cells are completely deposited. The preliminarily purified empty capsid antigen of rabbit hemorrhagic disease virus was still positive when diluted to 100,000 times.

(3) Electron microscope and colloidal gold immune electron microscope observation

After the collection and purification of virus-like particles, the sample was diluted 10 times with water, put on a copper grid, the filter paper was blotted dry, washed with ddH ₂ O, and then blotted dry with a filter paper, and the copper grid was placed on the uranyl acetate droplet. After absorbing the liquid on the filter paper sheet, put it on a new uranyl acetate droplet, and observe it with an electron microscope after repeating it 3 times, as shown in Figure 1, the empty capsid particles of rabbit hemorrhagic fever virus can be observed, and the size is consistent with the expectation. After diluting the sample 40 times with water, put it on the copper grid, blot the liquid on the filter paper, wash it with ddH ₂ O, then blot it dry with the filter paper, place the copper grid on the diluted primary antibody drop for about 3 minutes, drip wash and blot dry Put it on the diluted colloidal gold secondary antibody droplet, drip wash and blot dry, place the copper grid on the uranium acetate droplet, wash and dye for 3 times and then observe with the electron microscope, as shown in Figure 2, the rabbit hemorrhagic fever virus adsorbed on colloidal gold can be observed The number of empty capsid particles is quite large, indicating that the rabbit hemorrhagic fever virus protein VP60 can be assembled into virus empty capsid particles in the silkworm body independently.

(4) Animal immunization experiments

The experimental procedure is the same as that in Example 1. The collected purified antigen RHDV virus capsid particles are immunized by intramuscular injection and oral route respectively to 10 rabbits as the test group, and another 5 rabbits are only injected with adjuvant and fed with the antigen as the control group. , Rabbits are RHDV negative. 5 micrograms of RHDV empty capsid antigen and oil adjuvant were mixed in equal volumes to make a trial vaccine, 1ml/only for animal experiments on rabbits, and blood was collected after 21 days for ELISA antibody titer detection. All the test groups produced antibodies against RHDV. The detection titer of the injection group can reach more than 3200 times; the initially purified empty capsid virus particles are mixed with the same volume of propolis containing 25%-35% fatty acid and 8%-12% according to the amount of 50 μg per rabbit, The optimal concentration is 30% fatty acid and 10% propolis (fatty acid composition is palmitic acid 8%, oleic acid 19%, other components are 3%), perfusion after ultrasonic emulsification, one month later, the IgA titer of oral group detection also reaches 1600 times. The results showed that 100% of the test group produced protective antibodies, but no antibodies were detected in the control group; the challenge experiment was carried out at the same time as blood collection, and no rabbits in the immune group died no matter whether they were injected or orally administered, while all rabbits in the control group died within 3 days after challenge. Typical symptoms of rabbit hemorrhagic fever.

Claims (10)

1. A rabbit hemorrhagic fever virus capsid protein VP60 optimization gene is characterized in that its nucleotide sequence is as shown in SEQ ID No.2. 2. A recombinant baculovirus, characterized in that, by using the baculovirus transfer vector DNA and the baculovirus DNA comprising the rabbit hemorrhagic fever virus capsid protein VP60 gene or the optimized gene according to claim 1 to carry out recombination or transposition obtained after the reaction. 3. The recombinant baculovirus according to claim 2, characterized in that, the recombinant baculovirus is the recombinant silkworm nucleopolyhedrosis virus rBmNPV comprising the rabbit hemorrhagic fever virus capsid protein VP60 gene, or comprising the recombinant baculovirus of claim 1 The recombinant silkworm nuclear polyhedrosis virus rBmNPV of the optimized gene. 4. an insect bioreactor for preparing rabbit hemorrhagic fever virus capsid protein VP60 is characterized in that, by using the recombinant baculovirus infection insect of expressing rabbit hemorrhagic fever virus capsid protein VP60 gene or the optimized gene described in claim 1 obtained by the host. 5. a preparation method of rabbit hemorrhagic fever virus empty capsid antigen, is characterized in that, described method comprises the following steps: 1) constructing a baculovirus transfer vector comprising the rabbit hemorrhagic fever virus capsid protein VP60 gene or the optimized gene described in claim 1; 2) co-transfecting the constructed transfer expression vector with baculovirus to obtain recombinant baculovirus; 3) Infecting insect host cells with the recombinant baculovirus; 4) culturing the infected insect host, expressing the corresponding rabbit hemorrhagic fever virus capsid antigen particles, harvesting and purifying the expressed antigen. 6. The method according to claim 5, wherein the baculovirus is selected from BmNPV, AcMNPV, ApNPV, HaNPV, HzNPV, LdMNPV, MbMNPV, OpMNPV, SlMNPV, SeMNPV or SpltNPV, preferably the silkworm karyotype polygonal Somatic virus BmNPV. 7. method according to claim 5, is characterized in that, described host is selected from Lepidoptera insect, is preferably silkworm (Bombyx mori), wild silkworm (Bombyx mandarina), castor silkworm (Philosamia cynthia ricim), camphor Silkworm (Dicyoplca japanica), Chinese silkworm (Philosamia cynthia pryeri), tussah silkworm (Antheraea pernyi), Japanese tussah silkworm (Antheraea yamamai), wild silkworm (Antheraea polyphymus), alfalfa looper (Atographacaliforica), tea looper (Ectropis obliqua), cabbage night Moth (Mamestra brassicae), Spodoptera littoralis, Fall Armyworm (Spodoptera frugiperda), Trichoplusia ni, Armyworm (Thaumetopoea wilkinsoni), Cotton bollworm (Heliothis armigera), American bollworm (Heliothiszea ), tobacco budworm (Heliothis assulta), tobacco armyworm (Heliothis virescens), oriental armyworm (Pseudaletia separata) or gypsy moth (Lymantria dispar), more preferably silkworm. 8. The method of claim 5, wherein, The insect hosts are silkworm larvae and pupae. Infect silkworm cells with the recombinant silkworm baculovirus or puncture inoculate 1-5 instar silkworm larvae or pupae, and collect body fluid or tissue homogenate of silkworm larvae or pupae 3-6 days after infection . 9. The rabbit hemorrhagic fever virus capsid protein VP60 prepared by the method according to any one of claims 5-8. 10. The application of the rabbit hemorrhagic fever virus capsid protein VP60 as claimed in claim 9 as a rabbit hemorrhagic fever vaccine.

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