CN101307317A - Preparation method of rabies virus antigen - Google Patents

Abstract

The invention provides a method for preparing rabies virus antigen, comprising: respectively cloning the antigen gene of rabies virus or the joint expression combination of antigen genes into a baculovirus carrier to obtain a transfer expression carrier; combining the transfer expression carrier with a baculovirus Viruses are co-transfected for homologous recombination or transposition to obtain recombinant baculoviruses; insect hosts and cells are infected with recombinant baculoviruses; infected insect hosts are cultured to express corresponding rabies antigens; expressed antigens are harvested and purified , that is. The method of the invention adopts the baculovirus expression system to produce safe and efficient rabies virus antigen in the silkworm bioreactor, and because the prepared antigen has high safety, it can be directly used for producing injection and oral vaccines for immunizing animals. The method of the invention can greatly reduce the production cost of the rabies antigen, and has many advantages such as safety, high efficiency, less energy consumption, and low cost.

Description

Preparation method of rabies virus antigen

technical field

The invention relates to a method for preparing an antigen, in particular to a method for expressing a rabies virus antigen gene or combined antigen gene in insects by using a recombinant baculovirus, which is used for preparing injection and oral rabies vaccines and belongs to the field of genetic engineering.

Background technique

Rabies is a zoonotic and fatal encephalomyelitis caused by rabies virus (RV) invading the central nervous system. Once the disease occurs, the mortality rate is almost 100%. So far, there is no specific treatment drug, and the only effective way is to prevent it by vaccination. Therefore, large-scale preventive immunization to dogs, cats and wild animals is the fundamental measure to control and eliminate rabies. At present, the rabies vaccines for veterinary use in my country mainly include inactivated vaccines and attenuated vaccines. Although traditional vaccines still play a dominant role in the prevention and control of rabies, the production costs are high, the immunization period is short, and risk factors such as virus escape during vaccine preparation are difficult to avoid. Therefore, traditional vaccines need to be improved urgently, and it is still necessary to develop safe and efficient rabies molecular vaccines.

With the development of high and new technologies such as biotechnology and genetic engineering, people realize that by means of genetic engineering, using bioreactors to express rabies genes efficiently, it is expected to greatly increase the production of rabies antigens and reduce production costs (ZHEN FANG Fu, Immunology, vol.88, pp.2001-2005, March 1991,).

The bioreactor of the baculovirus expression system was established in the 1980s. Since the first high-efficiency expression of human α-interferon using the baculovirus expression system in 1983 (Smith, Mol. Cell Biol., 3: 2156-2165, 1983), hundreds of foreign genes have been highly expressed In China alone, there are empty capsid particles of foot-and-mouth disease (Li Zhiyong et al., Plos one, e2273, 2008), α-interferon (Yang Guanzhen et al., Acta Biochemistry and Biophysics, 22:355-361, 1990), arrowhead protease inhibition Agents (Ji Pingping, Sericulture Science, 21:223-227, 1995), Marek's virus glycoprotein B (Xiao Qingli et al., Sericulture Science, 23:104-108, 1997), etc. The advantages of using this system to produce rabies antigen are: 1. The expression efficiency of this expression system is extremely high, and the yield can reach the level of 10 mg/worm. Therefore, it can greatly reduce the production cost of rabies antigen and make it possible to produce rabies antigen on a large scale through genetic engineering; 2. This expression system is a eukaryotic expression system, and the foreign protein expressed can be post-translationally The biochemical properties and biological activities are similar to natural products, which guarantees that the expressed rabies antigen has normal protein structure and biological immune activity. At present, the baculovirus expression system, especially the Bombyx mori (Bm)-bombyx mori baculovirus (BmNPV) expression system is one of the most commercially exploitable eukaryotic individual expression systems in the world.

Contents of the invention

The technical problem to be solved by the present invention is to overcome the deficiencies of the prior art and provide a method for safely and efficiently expressing multiple rabies antigens in insects using a baculovirus expression system.

The technical problem to be solved by the present invention is achieved through the following technical solutions:

A method for preparing a rabies antigen, comprising: respectively cloning the antigen gene of rabies virus or the combined expression combination of antigen genes into a baculovirus carrier to construct a transfer expression vector; combining the constructed transfer expression vector with baculovirus DNA Carry out co-transfection for homologous recombination or transposition, transfer the antigenic gene of rabies virus or the joint expression combination of antigenic gene to the genome of baculovirus to obtain recombinant baculovirus; use recombinant baculovirus to infect insect host and cells; culturing infected insect hosts to express corresponding rabies antigens; harvesting and purifying the expressed antigens to obtain.

Wherein, the antigenic gene of described rabies virus is preferably rabies virus antigenic protein G gene (its nucleotide sequence is shown in SEQ ID NO: 1) or rabies virus antigenic protein N gene (its nucleotide sequence is SEQ ID NO : shown in 3); the combined expression combination of the antigenic gene of the rabies virus is the combined expression combination of the rabies virus antigenic protein G gene and the rabies virus antigenic protein antigenic protein N gene (i.e. the G-IRES-N combination gene), its nucleus The nucleotide sequence is shown in SEQ ID NO: 5;

The expression cassette of the baculovirus carrier vector is preferably an expression cassette composed of a polyhedrin promoter, p10 promoter or ie-1 promoter and an enhancer, for example, the baculovirus carrier vector can be selected from pBM034 , pBM93, AcRP23-lacZ, AcRP6-SC, AcUW1-lacZ, BacPAK6, Bac to Pac, Bacmid, BlucBacII (pETL), p2Bac, p2Blue, p89B310, pAc360, pAc373, pAcAB3, pAcAB4, PAcAS3, pAcC129, pAcC4, DZI ，pAcGP67，pAcIE1，pAcJP1，pAcMLF2，pAcMLF7，pAcMLF8，pAcMP1，pAcMP2，pAcRP23，pAcRP25，pAcRW4，pAcsMAG，pAcUW1，pAcUW21，pAcUW2A，pAcUW2B，pAcUW3，pAcUW31，pAcUW41，pAcUW42，pAcUW43，pAcUW51，pAcVC2，pAcVC3，pAcYM1 , pAcJcC5, pBac1, pBac2, pBlueBacIII, pBlueBacHis, pEV55, mXIV, pIEINeo, pJVETL, pJVNhel, pJVP10, pJVrsMAG, pMBac, pP10, pPAK1, pPBac, pSHONEX1.1, pSYN XIV VI+, pSYNVI+wp, pSYNXIVLVI , pVL 1392, pVL 1393, pVL941, pVL 945, pVL 985, pVTBac, pBM030, pUAC-5 or other similar baculovirus homologous recombination or transposable vectors, more preferably pVL1393 virus carrier vectors.

The transfer vector constructed in the present invention is preferably pVL1393(G), pVL1393(N), or pVL1393(G-IRES-N) (that is, using IRES (internal ribosome entry site, Chain A, Structure Of Ribosome-Bound Cricket Paralysis Virus Ires Rna.) sequence in the silkworm silkworm with a recombinant virus simultaneous dual-gene combined expression).

The baculovirus is preferably selected from BmNPV, AcMNPV, ApNPV, HaNPV, HzNPV, LdMNPV, MbMNPV, OpMNPV, SlMNPV, SeMNPV or SpltNPV, more preferably the parent strain of Bombyx mori baculovirus Bm-NPV-ZJ8.

Described recombinant baculovirus is preferably any one of the following: (1) recombinant silkworm nuclear polyhedrosis virus rBmNPV (G) for expressing rabies glycoprotein G; (its microorganism preservation number is: CGMCC No.2550; preservation Address: Institute of Microbiology, Chinese Academy of Sciences, Datun Road, Chaoyang District, Beijing; Preservation unit: General Microbiology Center of China Microbiological Culture Collection Management Committee; Preservation time: June 18, 2008; Classification and name: Bombyx mori nuclear polyhedrosis virus Bombyxmori Nucleopolyhedrovirus); (2) Recombinant Bombyx mori nucleopolyhedrovirus rBmNPV(N) for expressing rabies nucleoprotein N; (3) Recombinant Bombyx mori nucleopolyhedrovirus for simultaneous expression of rabies glycoprotein G and rabies nucleoprotein N Virus rBmNPV (G-IRES-N).

The insect host is selected from the group consisting of silkworm (Bombyx mori), wild silkworm (Bombyx mandarina), castor silkworm (Philosamia cynthia ricim), camphor silkworm (Dictyoploca japanica), Japanese silkworm (Philosamia cynthia pryeri), tussah silkworm (Antheraea pemyi), Antheraea yamamai, Antheraea polyphymus, Atographa califorica, Ectropis obliqua, Mamestra brassicae, Spodoptera littoralis, Spodoptera frugiperda), Trichoplusiani, Armyworm (Thaumetopoea wilkinsoni), Cotton bollworm (Heliothis armigera), American bollworm (Heliothiszea), Tobacco budworm (Heliothis assulta), Tobacco armyworm (Heliothis virescens), Oriental armyworm (Pseudaletia sparata), gypsy moth (Lymantria dispar), etc.; more preferably silkworm (Bombyx mori).

Described infection refers to that recombinant baculovirus infects 1-5 instar insect larvae or pupal bodies by swallowing or penetrating through the epidermis (more preferably: infecting silkworm cells with recombinant baculovirus or puncturing and inoculating 1-5 instar insect larvae or pupae) Bombyx mori larvae or pupae, body fluid or tissue homogenate of silkworm larvae or pupae containing various rabies antigens are collected 3-6 days after infection; wherein, the pupae body is an early tender pupae of 1-2 days.

The present invention uses gene recombination technology to construct different gene combinations derived from rabies virus, including glycoprotein G, nucleoprotein N, glycoprotein G and nucleoprotein N gene joint expression combination G-IRES-N of rabies into various gene combinations composed of The promoter is the baculovirus expression transfer carrier (such as AcRP23-lacZ, AcRP6-SC, AcUW1-lacZ, 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, pSYNXIVVI+, pSYNVI+wp, pSYNXIVVI-, pVL1391, 1392, 1393, pVL941, 945, 985, pVTBac, pUM035) , to make different gene combinations of rabies virus, including G, N or G-IRES-N gene under the control of polyhedron promoter, p10 promoter or other strong promoters of viruses and eukaryotes, through in vivo or in vitro ( in vivo/in vitro) recombination, different gene combinations of rabies virus, including G, N or G-IRES-N, are integrated into the genome of baculovirus to obtain recombinant virus; This method infects insect larvae or pupae of 1-5 instars (the optimal time is four or five instars) through the epidermis (the optimal time is early tender pupae of 1-2 days), and expresses and produces various rabies virus antigens.

A most preferred technical scheme in the present invention is: SEQ ID NO: 1 (rabies virus antigenic protein G gene), SEQ ID NO: 3 (rabies virus antigenic protein N gene) or SEQ ID NO: 5 (G-IRES- The DNA sequence shown in N) was inserted into the carrier vector pVL1393, and then the full-length rabies virus G, N or G-IRES-N gene was transferred to the genome of the silkworm baculovirus parent strain Bm-NPV-ZJ8 by recombination in vivo , replace the Polyhedrin gene on the genome, and obtain recombinant Bombyx mori baculovirus rBmNPV(G), rBmNPV(N), rBmNPV(G-IRES-N) carrying different gene combinations of rabies through plaque screening technology and PCR detection technology; It infects silkworm cell lines or punctures and inoculates 1-5 instar silkworm larvae or pupae to multiply rBmNPV(G), rBmNPV(N), rBmNPV(G-IRES-N); when rBmNPV(G), rBmNPV(N), When rBmNPV (G-IRES-N) replicates in silkworms, G, N or G-IRES-N genes are expressed under the control of the polyhedrin gene (polh) promoter to produce rabies antigens; It is best to collect the body fluid (or whole homogenate) of silkworm larvae or pupae containing rabies antigen after 5 days, 25 degrees feeding temperature), and each milliliter of silkworm hemolymph can produce more than 10 mg of corresponding rabies antigen, after killing the infectious pathogen , after protein purification, a safe and efficient rabies antigen can be obtained, which can be used to prepare an injection vaccine for preventing rabies; The concentration of volume is the fatty acid of 25-35% (in order to reach optimum effect, described fatty acid preferably is made up of each component of following volume percentage: palmitic acid 7%, oleic acid 20.5%, 3% stearic acid, The balance is water) mixed together, after ultrasonic emulsification, injection or oral vaccine can be obtained, and the animal can produce corresponding protective antibodies and can withstand the attack of rabies virus. This kind of antigen can be used to prepare and prevent rabies Oral vaccines.

The method of the present invention adopts the baculovirus expression system to safely and efficiently produce the rabies antigen in the silkworm bioreactor, and its production cost is significantly lower than the traditional method for preparing the rabies antigen (such as preparing the rabies antigen by cell propagation virus), without investment and construction of a factory , no three wastes, very little energy consumption such as electricity and water resources. 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 invention can greatly reduce the production cost of the rabies antigen, and has many advantages such as safety, high efficiency, less energy consumption, and low cost.

Detailed ways

In order to further illustrate the present invention, a series of examples are given below. These examples are entirely illustrative, and they are only used to specifically describe the present invention, and should not be construed as limiting the present invention.

experiment material

1. Escherichia coli strains E.coli TG1 and DH _5α were purchased from Promega; carrier vector pVL1393 was purchased from Invitrogen; silkworm cell BmN and silkworm nuclear polyhedrosis virus parent strain Bm-NPV-ZJ8 were purchased from the Biotechnology Research Institute of the Chinese Academy of Agricultural Sciences The rabies virus was preserved by the Infectious Disease Laboratory of Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences; the antigen detection kit was purchased from the Genetic Engineering Laboratory of Wuhan Institute of Biological Products, and the highly expressed silkworm variety JY1 was preserved by the Institute of Biotechnology, Chinese Academy of Agricultural Sciences.

2. Enzymes and reagents: Restriction enzymes, PNK enzymes, and ligases are products of Promega.

3. Biochemical reagents: IPTG and X-Gal are products of Promega. Lipofectin, low melting point agarose LMP, PCR kit, _T4 DNA ligase, RNase, Proteinase K, fetal bovine serum and other reagents were purchased from Invitrogen Company, and cell culture medium TC-100 was purchased from Sigma Company.

4. Culture medium: Escherichia coli culture medium is LB (1% peptone, 0.5% yeast extract, 1% NaCl, pH7.0); silkworm cell culture medium is TC-100.

5. Animal experiments on the expression products of different combinations of rabies virus genes were carried out in the animal isolation laboratory of Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences.

Example 1 Preparation and purification of rabies antigen G protein, animal immunization experiment and virus challenge protection experiment

1. Cloning and sequence analysis of rabies virus antigen protein G gene

1.1 Obtaining the target gene

Primers were designed to amplify the rabies virus antigen protein G gene (SEQ ID NO: 1) by RT-PCR.

The amplification primers of the designed antigenic protein G gene are:

5′ upstream of pVL-G A GGATCC AACATGGTTCCTCAGGTTCTT 3′

Bam H I

Downstream of pVL-G 5′A GAATTC TCACAGTCTGATCTCACC 3′

EcoR I

Take a mouse that was killed by rabies virus CVS strain 90h, and aseptically dissect the brain tissue, and grind it according to the conventional method. After grinding, make a 1:5 suspension with cell culture medium (MEM), add an appropriate amount of antibiotics, soak the poison at room temperature for 6 hours or overnight at 4°C, and then freeze and thaw several times to lyse the cells. Centrifuge at 7500r/min for 5min, and take the supernatant for the extraction of total RNA.

Total RNA was extracted from the brain tissue of mice killed by rabies virus infection. The extracted total RNA was reverse-transcribed at 42°C to prepare cDNA using the PVL-G upstream primer under the action of AMV reverse transcriptase. The obtained cDNA was used as a template, and PCR amplification was carried out with specific primers.

The PCR reaction system is as follows:

Table 1 PCR reaction conditions

PCR reaction process: Denaturation at 94°C for 10 minutes; 1 minute at 94°C, 1 minute at 59°C, 2 minutes at 72°C, a total of 30 cycles. The final extension reaction was 10 min.

1.2. Purification of PCR products

The G gene product amplified by PCR was subjected to 1% agarose gel electrophoresis, and it was found that a fragment of about 1.5 kb was amplified. Cut the gel containing the corresponding DNA fragments with a sterilized scalpel under ultraviolet light, and then use the Geneclean kit for purification. The method is as follows: cut out the gel fragment and weigh it, put it into a sterilized 1.5ml small centrifuge tube, add 3 times (v/w) volume of 6M NaI, dissolve the gel at 37°C, add 10μl glass milk (Glass milk), mix well and place at room temperature for 5 minutes to make the DNA fully adsorbed on the glass milk, centrifuge at 12000rpm for 5 seconds, then wash with New Wash solution three times, each time the precipitate is bounced and centrifuged. Finally, after drying the precipitate, add 30 μl 0.1×TE Buffer to dissolve the DNA, centrifuge to remove the precipitate, and take the supernatant for further analysis.

1.3. Digestion and ligation reactions

Enzyme digestion reaction: The purified G fragment was analyzed by double digestion with BamHI and EcoRI. The total reaction volume was 50 μl, including 10 μl of purified PCR product, 5 μl of 10×enzyme corresponding buffer, 1 μl of each of the two enzymes, and supplemented with sterile water. volume. React at 37°C for more than 2 hours. The transfer plasmid pGEM-3Z was subjected to the same digestion reaction. Inactivate at 65°C for 10 minutes after the reaction.

Ligation reaction: total ligation volume 15 μl, PCR product 8 μl, carrier 1 μl, 5×T4 DNA ligation buffer 3 μl, T4 DNA ligase 1 μl, sterile water to make up volume, 12-14°C overnight for ligation.

1.4. Genetic transformation of Escherichia coli

Prepare E. coli TG1 competent cells with 75 mM CaCl ₂ . Take 5 μl of the ligation mixture prepared in step 3, add it to 200 μl competent cells, mix gently, bathe in ice for 30 minutes, heat shock at 42°C for 2 minutes, quickly put it on ice for 1-2 minutes, add the cells that have been incubated to 37°C 500 μl of LB medium, cultured at 37°C for 1 hour, 100-200 μl was spread on LB solid medium plate containing 100 μg/ml ampicillin (Amp), and cultured upside down at 37°C overnight.

1.5. Preparation of plasmid DNA

(1) Pick a single colony from the transformed LB plate, inoculate it in 3ml LB medium containing 100μg/ml Amp, and culture overnight at 37°C.

(2) Take 1.5ml of bacterial liquid in a small centrifuge tube, centrifuge at 3500rpm for 4min, and remove the supernatant.

(3) Add 150 μl of Solution I, mix well and place on ice at 15°C.

(4) Add 300 μl of Solution II and 150 μl of chloroform, mix gently and let stand for 5 minutes.

(5) Add 450 μl of Solution III, mix well and place on ice for 15 minutes.

(6) Centrifuge at 11000g for 10min, and transfer the supernatant to a new tube.

(7) Add 450 μl of isopropanol, mix well and place at 4°C for 15 minutes.

(8) Centrifuge at 11000 g for 6 min, and remove the supernatant.

(9) Add 250 μl of TER, mix well and place at 37°C for 20 minutes.

(10) Add 300-350 μl of PPt Buffer, mix well and let stand for 15 minutes.

(11) Centrifuge at 11000 g for 6 min, and remove the supernatant.

(12) Add 400 μl of 75% ethanol.

(13) Centrifuge at 11000g for 3min, pour off ethanol, drain and add 40μl of 0.1×TE Buffer to dissolve, and store at -20°C.

1.6. Identification of recombinants

Digest the plasmid DNA prepared in step 5 with BamHI/EcoRI, and the plasmid with a DNA band of about 1.5 kb after electrophoresis is the recombinant carrier plasmid pGEM-3Z(G). The recombinant plasmid pGEM-3Z(G) was subjected to bidirectional sequencing. The G gene sequence and the deduced amino acid sequence are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively.

2. Construction of transfer vector pVL1393(G)

Digest the plasmid pGEM-3Z(G) prepared in 1.6 with BamHI and EcoRI, purify the G gene fragment by the method in 1.2, connect it with the baculovirus transfer vector pVL1393 that has been digested with BamHI and EcoRI, and transform the large intestine Bacillus TG1 was screened to obtain the recombinant transfer vector pVL1393(G).

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

Prepare 1 × TC-100 medium according to the product instructions of GIBCO company, adjust the pH to 6.22 with 2N NaOH, add 10% fetal bovine serum to the filter-sterilized medium, and cultivate silkworm cell BmN 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 (5000rpm×10min), remove the precipitate, and supernatant Centrifuge at 25,000rpm for 1 hour, remove the supernatant, suspend the virus particle pellet with 1ml virus DNA extract (1000ml contains 12.1g Tris, 33.6g EDTA, 14.1g KCl, pH7.5), transfer to a 1.5ml centrifuge tube, Add proteinase K to a final concentration of 50 μg/ml, incubate at 50°C for 2 hours, then add 35% Sarkorsel to a final concentration of 1%, continue to incubate at 50°C for 2 hours, and use equal volumes of phenol and phenol:chloroform (1 : 1) Chloroform extraction in sequence, transfer the upper aqueous phase to a new tube, add 1/10 volume of 3M NaCl, then add 2 times the volume of absolute ethanol, place at -20°C for more than 2 hours to precipitate virus DNA, 5000rpm After centrifugation for 10 min, the precipitate was washed once with 75% ethanol and freeze-dried. Dissolve in 100μl TE Buffer and store at 4°C for later use.

4. Construction and acquisition of recombinant Bombyx mori baculovirus rBmNPV(G)

4.1 Construction of recombinant baculovirus rBmNPV(G)

Inoculate approximately 1×10 ⁶ cells in a 15 cm ² 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 1.5 ml of medium without FBS. Add 1 μg of silkworm baculovirus parent strain Bm-NPV-ZJ8 DNA, 2 μg of recombinant transfer plasmid pVL1393(G) DNA and 5 μl of liposomes to a sterilized tube in sequence, make up the volume to 60 μl with sterile double distilled water, and gently Mix well, let it stand for 15 minutes, and add it dropwise into the culture bottle for co-transfection. After culturing at 27° C. for 4 hours, 1.5 ml serum-free medium and 300 μl FBS were added. Culture at a constant temperature of 27° C. for 4 to 5 days, and collect the supernatant for screening of recombinant viruses.

4.2 Screening and purification of recombinant Bombyx mori baculovirus rBmNPV(G)

Inoculate an appropriate amount of cells (about 70-80%) in a 35mm small plate. After the cells adhere to the wall, suck off the medium, dilute the co-transfection supernatant at different concentrations, take 1ml of the co-transfection solution and add it to the adherent cells. evenly distributed. After infection at 27°C for 1 hour, suck out the infection solution, melt 2% low-melting point agarose gel in a 60°C water bath, cool to 40°C and preheat 2×TC-100 medium (containing 20% FBS) at 40°C ) and mix evenly, add 4ml of glue to each plate, seal with Parafilm after solidification, incubate upside down at 27°C for 3-5 days, and observe under a microscope. The plaques without polyhedrons were selected, and the above steps were repeated to obtain pure recombinant Bombyx mori baculovirus rBmNPV(G) through 2-3 rounds of purification.

4.3 Amplification of recombinant virus rBmNPV(G) in silkworm cells

The recombinant Bombyx mori baculovirus rBmNPV(G) was used to infect normal growing BmN cells, and the supernatant was collected after culturing for 3 days, and the supernatant contained a large amount of recombinant virus rBmNPV(G).

4.4 Identification of recombinant virus

The foreign gene integration was analyzed by PCR method. The extraction method of free viral genomic DNA is as follows: take 150 μl of viral supernatant, add 150 μl (0.5mol/L) of NaOH and mix well, then add 20 μl (8mol/L) of ammonium acetate, mix well and use an equal volume of phenol and Chloroform was extracted once respectively, and after alcohol precipitation, 20 μl of TE was used to dissolve DNA. The oligonucleotide primers are:

5'-GAGGATCCACGATGAAAGCGATCTTAATCCCAT-3'

5'-AGAATTCTCACAGTCTGATCTCACC-3'

Take 1 μl of the above viral genome DNA for PCR amplification, the reaction conditions are: denaturation at 94°C for 5 minutes, 1 minute at 94°C, 1 minute at 58°C, 1 minute at 72°C, 30 cycles, and finally extension at 72°C for 5 minutes. 15 μl of the reaction product was analyzed by electrophoresis, and the result proved that the recombinant virus was obtained.

5. G gene is highly expressed in silkworm chrysalis and silkworm body

The silkworm chrysalis used in this experiment was JY1 (preserved by our laboratory) with high expression. 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. Seven days after cocooning, 15 silkworm chrysalis with the same average body weight were selected, and each silkworm chrysalis and silkworm were inoculated with about 1.0×10 ⁵ rBmNPV(G). After 4-5 days, the diseased silkworm chrysalis and silkworm blood were collected and stored at -20°C for double antibody Sandwich ELISA assay. From the results of OD value determination experiments, the expression level reached more than 10 times that of the positive control traditional cell vaccine virus antigen expression level, while no antigen expression was detected in the silkworm hemolymph infected with control Bm-NPV-ZJ8.

6. Collection of rabies virus G antigen

Since rabies virus G protein is a membrane protein, purification is difficult. Therefore, the silkworm hemolymph harvested in the above 5 was ultrasonically disrupted, centrifuged to remove cell debris, and then irradiated to obtain sterile antigen, and the G antigen was further purified by chromatography and other methods.

7. Animal immunity experiment and virus attack protection experiment

Mix the collected and purified sterile antigen with an equal volume of oil adjuvant to prepare a trial vaccine, and conduct animal experiments on mice. 0.5ml/only to immunize 10 mice through intramuscular or oral routes, and another 5 mice were not injected with anything as the control group. One month later, the standard challenge strain of lethal rabies virus CVS was challenged in the brain, and as a result, the test group obtained 90% protection, while all the control group became ill.

The rabies virus G antigen of primary purification is 30.5% fatty acid (this fatty acid is made up of each component of following volume percentage with equal volume concentration: palmitic acid 7%, oleic acid 20.5%, 3% stearic acid, balance is water ) mixed, prepared into an oral vaccine after phacoemulsification, perfused through the mouth, and after one month, challenged with the lethal rabies virus CVS standard challenge strain in the brain. As a result, the oral test group obtained 80% protection, while all the control groups were ill. .

Whether the antigen prepared in this example was injected or orally administered, 100% of the animals produced protective antibodies.

Example 2 Preparation and purification of rabies antigen N protein, animal immunization experiment and virus challenge protection experiment

1. Cloning and sequence analysis of rabies virus N gene

Primers were designed to amplify the rabies virus N gene (SEQ ID NO: 3) by RT-PCR.

The primers designed to amplify the N gene are:

Upstream of pVL-N 5′ A TCTAGA AACATGGATGCCGACAAGATT 3′

Xba I

Downstream of pVL-N 5′ A GCGGCCGC TTATGAGTCATTCGAATACGT 3′

Not I

Total RNA was extracted from the brain tissue of mice killed by rabies virus infection. The extracted total RNA was reverse-transcribed at 42°C to prepare cDNA using PVL-N upstream primers under the action of AMV reverse transcriptase. The obtained cDNA was used as a template, and PCR amplification was carried out with specific primers.

The PCR reaction system is as follows:

Table 2 PCR reaction conditions

PCR reaction process: Denaturation at 94°C for 10 minutes; 1 minute at 94°C, 1 minute at 56°C, 2 minutes at 72°C, a total of 30 cycles. The final extension reaction was 10 min.

2. Construction of transfer vector pVL1393(N)

The N amplified in 1 was purified according to the method of 1.2 in Example 1, after being digested with XbaI and NotI, it was ligated with the baculovirus transfer vector pVL1393 that had been digested with XbaI and NotI, and transformed into Escherichia coli TG1, and screened The recombinant transfer vector pVL1393(N) was obtained. The recombinant plasmid pVL1393(N) was subjected to bidirectional sequencing. The N gene sequence and the deduced amino acid sequence are shown in SEQ ID NO: 3 and SEQ ID NO: 4, respectively.

3. Construction and acquisition of recombinant Bombyx mori baculovirus rBmNPV(N)

3.1. Construction of recombinant baculovirus rBmNPV(N)

Inoculate approximately 1×10 ⁶ cells in a 15 cm ² culture flask. After the cells adhere to the wall, remove the fetal bovine serum (FBS) medium, wash with FBS-free medium three times, and add 1.5ml of FBS-free medium. Add 1 μg of silkworm baculovirus parent strain Bm-NPV-ZJ8 DNA, 2 μg of recombinant transfer plasmid pVL1393(N) DNA and 5 μl of liposomes to a sterilized tube in sequence, make up the volume to 60 μl with sterile double distilled water, and gently Mix well, let it stand for 15 minutes, and then add it dropwise into the culture flask for co-transfection. After culturing at 27° C. for 4 hours, 1.5 ml serum-free medium and 300 μl FBS were added. Culture at a constant temperature of 27° C. for 4 to 5 days, and collect the supernatant for screening of recombinant viruses.

3.2 Screening and purification of recombinant Bombyx mori baculovirus rBmNPV(N)

Inoculate an appropriate amount of cells (about 70-80%) in a 35mm small plate. After the cells adhere to the wall, suck off the medium, dilute the co-transfection supernatant at different concentrations, take 1ml of the co-transfection solution and add it to the adherent cells. evenly distributed. After infection at 27°C for 1 hour, suck out the infection solution, melt 2% low-melting point agarose gel in a 60°C water bath, cool to 40°C and preheat 2×TC-100 medium (containing 20% FBS) at 40°C ) and mix evenly, add 4ml of glue to each plate, seal with Parafilm after solidification, incubate upside down at 27°C for 3-5 days, and observe under a microscope. The plaques without polyhedrons were selected, and the above steps were repeated to obtain pure recombinant Bombyx mori baculovirus rBmNPV(N) through 2-3 rounds of purification.

3.3 Amplification of recombinant virus rBmNPV(N) in silkworm cells

The recombinant Bombyx mori baculovirus rBmNPV(N) was used to infect normal growing BmN cells, and after culturing for 3 days, the supernatant was collected, and the supernatant contained a large amount of recombinant virus rBmNPV(N).

3.4 Identification of recombinant virus

The foreign gene integration was analyzed by PCR method. The extraction method of free viral genomic DNA is as follows: take 150 μl of viral supernatant, add 150 μl (0.5mol/L) of NaOH and mix well, then add 20 μl (8mol/L) of ammonium acetate, mix well and use an equal volume of phenol and Chloroform was extracted once respectively, and after alcohol precipitation, 20 μl of TE was used to dissolve DNA. The oligonucleotide primers are:

5'-GAGGATCCACGATGAAGCGATCTTAATCCCAT-3'

5′AGCGGCCGCTTATGAGTCATTCGAATACGT 3′

Take 1 μl of the above viral genome DNA for PCR amplification, the reaction conditions are: denaturation at 94°C for 5 minutes, 1 minute at 94°C, 1 minute at 55°C, 1 minute at 72°C, 30 cycles, and finally extension at 72°C for 5 minutes. 15 μl of the reaction product was analyzed by electrophoresis, and the result proved that the recombinant virus was obtained.

4. N gene is expressed in silkworm body and pupa body

The silkworm chrysalis used in this experiment was JY1 (preserved by our laboratory) with high expression. 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. Seven days after cocooning, 15 silkworm chrysalis with the same average body weight were selected, and each silkworm chrysalis or silkworm was inoculated with about 1.0×10 ⁵ rBmNPV(N). After 4-5 days, the diseased silkworm chrysalis and silkworm blood were collected and stored at -20°C for double-antibody sandwiching ELISA method detection. From the results of OD value determination experiments, the expression level reached more than 100 times that of the positive control traditional cell vaccine virus antigen expression level, while no antigen expression was detected in the silkworm hemolymph infected with control Bm-NPV-ZJ8.

5. Antigen purification

Weigh the Sephadex xerogel, install it in a glass chromatographic column after swelling treatment, and wash it with eluent (5mmol/L Tris solution, 0.1mol/LNaCl, pH 8.0) until the baseline is stable. The 4 harvested silkworm hemolymph were ultrasonically disrupted and centrifuged to remove cell debris. Load the above sample, take an appropriate amount of eluent, flow rate 0.3ml/min, collect the protein eluate for 5min/tube, and collect until the first peak drops to obtain the purified antigen.

6. Animal immunity experiment and virus attack protection experiment

The collected purified antigen was mixed with an equal volume of oil adjuvant to prepare a trial vaccine, and animal experiments were carried out on mice. 0.5ml/only to immunize 10 mice through intramuscular or oral routes, and another 5 mice were not injected with anything as the control group. One month later, the standard challenge strain of lethal rabies virus CVS was challenged in the brain, and as a result, the test group obtained 70% protection, while the control group all became ill.

The rabies virus N antigen of initial purification is the fatty acid (this fatty acid is made up of each component of following volume percentage with the concentration of equal volume: palmitic acid 5%, oleic acid 18%, 2% stearic acid, balance is water) mixed, prepared into an oral vaccine after ultrasonic emulsification, perfused through the mouth, and after one month, challenged with the lethal rabies virus CVS standard challenge strain through the brain. As a result, the oral test group obtained 50% protection, while the control group had all sickness.

80-90% of animals with the antigen prepared in this example produced protective antibodies no matter whether they were injected or orally administered.

Example 3 Joint expression, purification, animal immunization experiment and virus challenge protection experiment of rabies virus G and N antigen

1. Cloning and sequence analysis of rabies virus G-IRES-N group and gene

Primers were designed to amplify the rabies virus G gene and N gene by RT-PCR.

The designed amplification primers for amplifying G and N genes are the same as before.

A section of IRES sequence that connects between G gene and N gene, with reference to the IRES of Rickettsia virus Cricket Paralysis Virus (internal ribosome entry site, Chain A, Structure Of Ribosome-Bound Cricket Paralysis Virus Ires Rna. See sequence SEQ ID NO6 ) sequence was artificially synthesized by our laboratory, and Xba I and Bgl II restriction sites were added to its 5' end and 3' end, respectively.

Total RNA was extracted from the brain tissue of mice killed by rabies virus infection. Using the upstream primers of pVL-G and pVL-N, the extracted total RNA was reverse-transcribed at 42°C under the action of AMV reverse transcriptase to prepare cDNA. The obtained amplified G gene and N gene cDNA were used as templates, and specific primers were used for PCR amplification, and the amplification conditions were the same as before.

2. Construction of transfer vector pVL1393(G-IRES-N)

Purify the G gene obtained by the method of 1.2 in Example 1, purify the N gene obtained by 1 and synthesize the IRES fragment by the method of 2.1 in Example 2, after digestion, connect with the baculovirus transfer vector pVL1393, and then transform into Escherichia coli TG1 was screened to obtain recombinant transfer carrier pVL1393 (G-IRES-N). The recombinant plasmid pVL1393(G-IRES-N) was sequenced bidirectionally. See SEQ ID NO:5 for the G-IRES-N gene sequence.

3. Construction and acquisition of recombinant silkworm baculovirus rBmNPV (G-IRES-N)

3.1 Construction of recombinant baculovirus rBmNPV (G-IRES-N)

Inoculate approximately 1×10 ⁶ cells in a 15 cm ² culture flask. After the cells adhere to the wall, remove the fetal bovine serum (FBS) medium, wash with FBS-free medium three times, and add 1.5ml of FBS-free medium. Add 1 μg of silkworm baculovirus parent strain Bm-NPV-ZJ8 DNA, 2 μg of recombinant transfer plasmid pVL1393 (G-IRES-N) DNA and 5 μl of liposomes to a sterilized tube, and make up the volume with sterile double distilled water to 60μl, mixed gently, let stand for 15 minutes, then added dropwise into the culture flask for co-transfection. After culturing at 27° C. for 4 hours, 1.5 ml serum-free medium and 300 μl FBS were added. Culture at a constant temperature of 27° C. for 4 to 5 days, and collect the supernatant for screening of recombinant viruses.

3.2 Screening and purification of recombinant Bombyx mori baculovirus rBmNPV (G-IRES-N)

Inoculate an appropriate amount of cells (about 70-80%) in a 35mm small plate. After the cells adhere to the wall, suck off the medium, dilute the co-transfection supernatant at different concentrations, take 1ml of the co-transfection solution and add it to the adherent cells. evenly distributed. After infection at 27°C for 1 hour, suck out the infection solution, melt 2% low-melting point agarose gel in a 60°C water bath, cool to 40°C and preheat 2×TC-100 medium (containing 20% FBS) at 40°C ) and mix evenly, add 4ml of glue to each plate, seal with Parafilm after solidification, incubate upside down at 27°C for 3-5 days, and observe under a microscope. The plaques without polyhedrons were selected, and the above steps were repeated to obtain pure recombinant Bombyx mori baculovirus rBmNPV (G-IRES-N) through 2-3 rounds of purification.

3.3 Amplification of recombinant virus rBmNPV (G-IRES-N) in silkworm cells

The recombinant Bombyx mori baculovirus rBmNPV (G-IRES-N) 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 (G-IRES-N).

3.4 Identification of recombinant virus

The foreign gene integration was analyzed by PCR method. The extraction method of free viral genomic DNA is as follows: take 150 μl of virus supernatant, add 150 μl (0.5mol/L) of NaOH and mix well, then add 20 μl (8mol/L) of ammonium acetate, mix well and use an equal volume of phenol and Chloroform was extracted once respectively, and after alcohol precipitation, 20 μl of TE was used to dissolve DNA. The oligonucleotide primers are:

5'-GAGGATCCACGATGAAAGCGATCTTAATCCCAT-3'

5′AGCGGCCGCTTATGAGTCATTCGAATACGT 3′

Take 1 μl of the above viral genomic DNA for PCR amplification, the reaction conditions are: denaturation at 94°C for 5 minutes, 1 minute at 94°C, 1 minute at 55°C, 1 minute at 72°C, 30 cycles, and finally extension at 72°C for 5 minutes. 15 μl of the reaction product was analyzed by electrophoresis, and the result proved that the recombinant virus was obtained.

4. Expression of G-IRES-N gene in silkworm pupae

The silkworm chrysalis used in this experiment was JY1 (preserved by our laboratory) with high expression. 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. Seven days after cocooning, 15 silkworm chrysalis with the same average body weight were selected, and each silkworm was inoculated with about 1.0×10 ⁵ rBmNPV (G-IRES-N). After 4-5 days, the diseased silkworm chrysalis were collected and stored at -20°C for double-antibody sandwich ELISA law detection. From the results of the OD value measurement experiment, the G antigen and N antigen expressed by it reached 15 times and 50 times the expression level of the positive control traditional cell vaccine virus antigen, while there was no expression in the silkworm hemolymph infected by the control Bm-NPV-ZJ8. Antigen expression detected.

5. Antigen collection

Since rabies virus G protein is a membrane protein, purification is difficult. Therefore, the silkworm hemolymph harvested in the above 4 was ultrasonically disrupted, centrifuged to remove cell debris, and then irradiated to obtain sterile antigens. For the purification of the co-expressed G and N proteins, refer to the purification section of the G and N proteins mentioned above, respectively.

6. Animal immunity experiment and virus attack protection experiment

Mix the collected and initially purified sterile antigen with an equal volume of oily complete adjuvant to prepare a trial vaccine, and conduct animal experiments on mice. 0.5ml/only to immunize 10 mice through intramuscular or oral routes, and another 5 mice were not injected with anything as the control group. One month later, the standard challenge strain of lethal rabies virus CVS was challenged in the brain, and as a result, the test group obtained 100% protection, while all the control group developed the disease.

The rabies virus G and N antigens of preliminary purification are 30.5% fatty acid (this fatty acid is made up of each component of following volume percentage with equal volume concentration: palmitic acid 7%, oleic acid 20.5%, 3% stearic acid, remaining (amount of water) mixed, prepared into an oral vaccine after ultrasonic emulsification, perfused through the mouth, and after one month, challenged with the lethal rabies virus CVS standard challenge strain through the brain, as a result, the oral test group obtained 100% protection, while the control All groups were affected.

It can be seen that the antigen prepared in this example produces high-titer protective antibodies in 100% of the animals whether injected or orally administered.

sequence listing

<110> Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences Institute of Biotechnology, Chinese Academy of Agricultural Sciences

<120> Preparation method of rabies virus antigen

<130>KLPI0786

<160>7

<170>PatentIn version 3.5

<210>1

<211>1575

<212>DNA

<213>Rabies Virus

<400>1

atggttcctc aggttctttt gtttgtactc cttctgggtt tttcgttgtg tttcgggaag 60

ttccccattt acacgatacc agacgaactt ggtccctgga gccctattga catacaccat 120

ctcagctgtc caaataacct ggttgtggag gatgaaggat gtaccaacct gtccgagttc 180

tcctacatgg aactcaaagt gggatacatc tcagccatca aagtgaacgg gttcacttgc 240

acagggtgttg tgacagaggc agagacctac accaactttg ttggttatgt cacaaccaca 300

ttcaagagaa agcatttccg ccccacccca gacgcatgta gagccgcgta taactggaag 360

atggccggtg accccagata tgaagagtcc ctacacaatc cataccccga ctaccactgg 420

cttcgaactg taagaaccac caaagagtcc ctcattatca tatccccaag tgtgacagat 480

ttggacccat atgacaaatc ccttcactca agggtcttcc ctggcggaaa gtgctcagga 540

ataacggtgt cctctaccta ctgctcaact aaccatgatt aaccatttg gatgcccgag 600

aatccgagac caaggacacc ttgtgacatt tttaccaata gcagagggaa gagagcatcc 660

aacgggaaca agacttgcgg ctctgtggat gaaagaggcc tgtataagtc tctaaaagga 720

gcatgcaggc tcaagttatg tggagttctt ggacttagac ttatggatgg aacatgggtc 780

gcgatgcaaa catcagatga gaccaaatgg tgccctccag atcagttggt gaatttgcac 840

gactttcgct cagacgagat tgagcatctc gttgtggagg agttagtcaa gaaaagagag 900

gaatgtctgg atgcattaga gtccatcatg accaccaagt cagtaagttt cagacgtctc 960

agtcacctga gaaaacttgt cccagggttt ggaaaagcat ataccatatt caacaaaacc 1020

ttgatggagg ctgatgctca ctacaagtca gtccggacct ggaatgagat catcccctca 1080

aaagggtgtt tgaaagttgg aggaaggtgc catcctcatg tgaacggggt gtttttcaat 1140

ggtataatat tagggcctga cgaccatgtc ctaatcccag agatgcaatc atccctcctc 1200

cagcaacata tggagttgtt ggaatcttca gttatccccc tgatgcaccc cctggcagac 1260

ccttctacag ttttcaaaga aggtgatgag gctgaggatt ttgttgaagt tcacctcccc 1320

gatgtgtaca aacagatctc aggggttgac ctgggtctcc cgaactgggg aaagtatgta 1380

ttgatgactg caggggccat gattggcctg gtgttgatat tttccctaat gacatggtgc 1440

agaagagcca atcgaccaga atcgaaacaa cgcagttttg gagggacagg ggggaatgtg 1500

tcagtcactt cccaaagcgg aaaagtcata ccttcatggg aatcatataa gagtggaggt 1560

gagatcagac tgtga 1575

<210>2

<211>524

<212>PRT

<213>Rabies Virus

<400>2

Met Val Pro Gln Val Leu Leu Phe Val Leu Leu Leu Gly Phe Ser Leu

1 5 10 15

Cys Phe Gly Lys Phe Pro Ile Tyr Thr Ile Pro Asp Glu Leu Gly Pro

20 25 30

Trp Ser Pro Ile Asp Ile His His Leu Ser Cys Pro Asn Asn Leu Val

35 40 45

Val Glu Asp Glu Gly Cys Thr Asn Leu Ser Glu Phe Ser Tyr Met Glu

50 55 60

Leu Lys Val Gly Tyr Ile Ser Ala Ile Lys Val Asn Gly Phe Thr Cys

65 70 75 80

Thr Gly Val Val Thr Glu Ala Glu Thr Tyr Thr Asn Phe Val Gly Tyr

85 90 95

Val Thr Thr Thr Phe Lys Arg Lys His Phe Arg Pro Thr Pro Asp Ala

100 105 110

Cys Arg Ala Ala Tyr Asn Trp Lys Met Ala Gly Asp Pro Arg Tyr Glu

115 120 125

Glu Ser Leu His Asn Pro Tyr Pro Asp Tyr His Trp Leu Arg Thr Val

130 135 140

Arg Thr Thr Lys Glu Ser Leu Ile Ile Ile Ser Pro Ser Val Thr Asp

145 150 155 160

Leu Asp Pro Tyr Asp Lys Ser Leu His Ser Arg Val Phe Pro Gly Gly

165 170 175

Lys Cys Ser Gly Ile Thr Val Ser Ser Thr Tyr Cys Ser Thr Asn His

180 185 190

Asp Tyr Thr Ile Trp Met Pro Glu Asn Pro Arg Pro Arg Thr Pro Cys

195 200 205

Asp Ile Phe Thr Asn Ser Arg Gly Lys Arg Ala Ser Asn Gly Asn Lys

210 215 220

Thr Cys Gly Ser Val Asp Glu Arg Gly Leu Tyr Lys Ser Leu Lys Gly

225 230 235 240

Ala Cys Arg Leu Lys Leu Cys Gly Val Leu Gly Leu Arg Leu Met Asp

245 250 255

Gly Thr Trp Val Ala Met Gln Thr Ser Asp Glu Thr Lys Trp Cys Pro

260 265 270

Pro Asp Gln Leu Val Asn Leu His Asp Phe Arg Ser Asp Glu Ile Glu

275 280 285

His Leu Val Val Glu Glu Leu Val Lys Lys Arg Glu Glu Cys Leu Asp

290 295 300

Ala Leu Glu Ser Ile Met Thr Thr Lys Ser Val Ser Phe Arg Arg Leu

305 310 315 320

Ser His Leu Arg Lys Leu Val Pro Gly Phe Gly Lys Ala Tyr Thr Ile

325 330 335

Phe Asn Lys Thr Leu Met Glu Ala Asp Ala His Tyr Lys Ser Val Arg

340 345 350

Thr Trp Asn Glu Ile Ile Pro Ser Lys Gly Cys Leu Lys Val Gly Gly

355 360 365

Arg Cys His Pro His Val Asn Gly Val Phe Phe Asn Gly Ile Ile Leu

370 375 380

Gly Pro Asp Asp His Val Leu Ile Pro Glu Met Gln Ser Ser Leu Leu

385 390 395 400

Gln Gln His Met Glu Leu Leu Glu Ser Ser Val Ile Pro Leu Met His

405 410 415

Pro Leu Ala Asp Pro Ser Thr Val Phe Lys Glu Gly Asp Glu Ala Glu

420 425 430

Asp Phe Val Glu Val His Leu Pro Asp Val Tyr Lys Gln Ile Ser Gly

435 440 445

Val Asp Leu Gly Leu Pro Asn Trp Gly Lys Tyr Val Leu Met Thr Ala

450 455 460

Gly Ala Met Ile Gly Leu Val Leu Ile Phe Ser Leu Met Thr Trp Cys

465 470 475 480

Arg Arg Ala Asn Arg Pro Glu Ser Lys Gln Arg Ser Phe Gly Gly Thr

485 490 495

Gly Gly Asn Val Ser Val Thr Ser Gln Ser Gly Lys Val Ile Pro Ser

500 505 510

Trp Glu Ser Tyr Lys Ser Gly Gly Glu Ile Arg Leu

515 520

<210>3

<211>1353

<212>DNA

<213> Rabies Virus

<400>3

atggatgccg acaagattgt gttcaaagtc aataatcagg tggtctcttt gaagcctgag 60

attatcgtgg atcaatatga gtacaagtac cctgccatca aggatttgaa aaagccttgt 120

atcaccctag ggaaagcccc cgacttgaac aaagcataca aatcagtttt atcaggcatg 180

aatgccgcca aacttgatcc ggatgatgta tgctcctact tggcagcagc aatgcagttc 240

tttgagggga catgtccgga agactggacc agctatggaa tcctgattgc acgaaaagga 300

gataggatca ccccaaactc tctagtggag ataaagcgta ctgatgtaga agggaattgg 360

gctctgacag gaggcatgga attgacaagg gaccccactg tctctgaaca tgcatcttta 420

gtcggtcttc tcctgagtct gtacaggttg agcaaaatat caggacagaa cactggtaac 480

tataagacaa acattgcaga taggatagag cagattttcg agacagcacc ttttgttaag 540

atcgtggaac accataccct aatgacaact cacaagatgt gtgctaattg gagtactata 600

ccgaacttca gatttttggc cggaacctac gacatgtttt tctcacggat tgagcatctg 660

tattcggcaa tcagagtggg cacagtcgtc accgcttatg aagactgctc aggactggta 720

tcgtttacag ggttcataaa gcagatcaat ctcaccgcaa gggaagcaat actatatttc 780

ttccacaaga actttgagga agagataaga agaatgttcg agccagggca agagacagct 840

gttcctcact cttatttcat ccacttccgt tcactaggct tgagtgggaa gtctccttat 900

tcatcgaatg ctgtcggtca tgtgttcaat ctcattcact ttgttggatg ctacatgggt 960

caagtcagat ctctaaatgc gacggttat gctgcatgtg cccctcatga gatgtctgtt 1020

ctagggggct atttgggaga ggaattcttc ggaaaaggga catttgaaag aaggttcttc 1080

agagacgaga aagaacttca agaatatgag gcggctgaac taacaaagtc cgacgtggca 1140

ctggcggatg acggaaccgt caactctgat gacgaggact atttctctgg tgaaaccaga 1200

agtccagaag ctgtctatac tcgaatcatg atgaatggag gtcgactgaa gagatctcat 1260

atacggagat atgtctcagt cagttccaat catcaagccc gtccaaactc attcgccgaa 1320

tttttaaaca agacgtattc gaatgactca taa 1353

<210>4

<211>450

<212>PRT

<213>Rabies Virus

<400>4

Met Asp Ala Asp Lys Ile Val Phe Lys Val Asn Asn Gln Val Val Ser

1 5 10 15

Leu Lys Pro Glu Ile Ile Val Asp Gln Tyr Glu Tyr Lys Tyr Pro Ala

20 25 30

Ile Lys Asp Leu Lys Lys Pro Cys Ile Thr Leu Gly Lys Ala Pro Asp

35 40 45

Leu Asn Lys Ala Tyr Lys Ser Val Leu Ser Gly Met Asn Ala Ala Lys

50 55 60

Leu Asp Pro Asp Asp Val Cys Ser Tyr Leu Ala Ala Ala Met Gln Phe

65 70 75 80

Phe Glu Gly Thr Cys Pro Glu Asp Trp Thr Ser Tyr Gly Ile Leu Ile

85 90 95

Ala Arg Lys Gly Asp Arg Ile Thr Pro Asn Ser Leu Val Glu Ile Lys

100 105 110

Arg Thr Asp Val Glu Gly Asn Trp Ala Leu Thr Gly Gly Met Glu Leu

115 120 125

Thr Arg Asp Pro Thr Val Ser Glu His Ala Ser Leu Val Gly Leu Leu

130 135 140

Leu Ser Leu Tyr Arg Leu Ser Lys Ile Ser Gly Gln Asn Thr Gly Asn

145 150 155 160

Tyr Lys Thr Asn Ile Ala Asp Arg Ile Glu Gln Ile Phe Glu Thr Ala

165 170 175

Pro Phe Val Lys Ile Val Glu His His Thr Leu Met Thr Thr His Lys

180 185 190

Met Cys Ala Asn Trp Ser Thr Ile Pro Asn Phe Arg Phe Leu Ala Gly

195 200 205

Thr Tyr Asp Met Phe Phe Ser Arg Ile Glu His Leu Tyr Ser Ala Ile

210 215 220

Arg Val Gly Thr Val Val Thr Ala Tyr Glu Asp Cys Ser Gly Leu Val

225 230 235 240

Ser Phe Thr Gly Phe Ile Lys Gln Ile Asn Leu Thr Ala Arg Glu Ala

245 250 255

Ile Leu Tyr Phe Phe His Lys Asn Phe Glu Glu Glu Ile Arg Arg Met

260 265 270

Phe Glu Pro Gly Gln Glu Thr Ala Val Pro His Ser Tyr Phe Ile His

275 280 285

Phe Arg Ser Leu Gly Leu Ser Gly Lys Ser Pro Tyr Ser Ser Asn Ala

290 295 300

Val Gly His Val Phe Asn Leu Ile His Phe Val Gly Cys Tyr Met Gly

305 310 315 320

Gln Val Arg Ser Leu Asn Ala Thr Val Ile Ala Ala Cys Ala Pro His

325 330 335

Glu Met Ser Val Leu Gly Gly Tyr Leu Gly Glu Glu Phe Phe Gly Lys

340 345 350

Gly Thr Phe Glu Arg Arg Phe Phe Arg Asp Glu Lys Glu Leu Gln Glu

355 360 365

Tyr Glu Ala Ala Glu Leu Thr Lys Ser Asp Val Ala Leu Ala Asp Asp

370 375 380

Gly Thr Val Asn Ser Asp Asp Glu Asp Tyr Phe Ser Gly Glu Thr Arg

385 390 395 400

Ser Pro Glu Ala Val Tyr Thr Arg Ile Met Met Asn Gly Gly Arg Leu

405 410 415

Lys Arg Ser His Ile Arg Arg Tyr Val Ser Val Ser Ser Asn His Gln

420 425 430

Ala Arg Pro Asn Ser Phe Ala Glu Phe Leu Asn Lys Thr Tyr Ser Asn

435 440 445

Asp Ser

450

<210>5

<211>3172

<212>DNA

<213>artificial sequence

<400>5

ggatccaaca tggttcctca ggttcttttg tttgtactcc ttctgggttt ttcgttgtgt 60

ttcgggaagt tccccatta cacgatacca gacgaacttg gtccctggag ccctattgac 120

atacaccatc tcagctgtcc aaataacctg gttgtggagg atgaaggatg taccaacctg 180

tccgagttct cctacatgga actcaaagtg ggatacatct cagccatcaa agtgaacggg 240

ttcacttgca caggtgttgt gacagaggca gagacctaca ccaactttgt tggttatgtc 300

acaaccacat tcaagagaaa gcatttccgc cccaccccag acgcatgtag agccgcgtat 360

aactggaaga tggccggtga ccccagatat gaagagtccc tacacaatcc atacccgac 420

taccactggc ttcgaactgt aagaaccacc aaagagtccc tcattatcat atccccaagt 480

gtgacagatt tggacccata tgacaaatcc cttcactcaa gggtcttccc tggcggaaag 540

tgctcaggaa taacggtgtc ctctacctac tgctcaacta accatgatta caccatttgg 600

atgcccgaga atccgagacc aaggacacct tgtgacattt ttaccaatag cagagggaag 660

agagcatcca acgggaacaa gacttgcggc tctgtggatg aaagaggcct gtataagtct 720

ctaaaaggag catgcaggct caagttatgt ggagttcttg gacttagact tatggatgga 780

acatgggtcg cgatgcaaac atcagatgag accaaatggt gccctccaga tcagttggtg 840

aatttgcacg actttcgctc agacgagatt gagcatctcg ttgtggagga gttagtcaag 900

aaaagagagg aatgtctgga tgcattagag tccatcatga ccaccaagtc agtaagtttc 960

agacgtctca gtcacctgag aaaacttgtc ccagggtttg gaaaagcata taccatattc 1020

aacaaaacct tgatggaggc tgatgctcac tacaagtcag tccggacctg gaatgagatc 1080

atcccctcaa aagggtgttt gaaagttgga ggaaggtgcc atcctcatgt gaacggggtg 1140

tttttcaatg gtataatatt agggcctgac gaccatgtcc taatcccaga gatgcaatca 1200

tccctcctcc agcaacatat ggagttgttg gaatcttcag ttatccccct gatgcacccc 1260

ctggcagacc cttctacagt tttcaaagaa ggtgatgagg ctgaggattt tgttgaagtt 1320

cacctccccg atgtgtacaa acagatctca ggggttgacc tgggtctccc gaactgggga 1380

aagtatgtat tgatgactgc aggggccatg attggcctgg tgttgatatt ttccctaatg 1440

acatggtgca gaagagccaa tcgaccagaa tcgaaacaac gcagttttgg agggacagggg 1500

gggaatgtgt cagtcacttc ccaaagcgga aaagtcatac cttcatggga atcatataag 1560

agtggaggtg agatcagact gtgatctaga aaagcaaaaa tgtgatcttg cttgtaaata 1620

caattttgag aggttaataa attacaagta gtgctatttt tgtatttagg ttagctattt 1680

agctttacgt tccaggatgc ctagtggcag ccccacaata tccaggaagc cctctctgcg 1740

gtttttcaga ttaggtagtc gaaaaaccta agaaatttac ctgctacatt tcaagatacc 1800

atggagatcc aacatggatg ccgacaagat tgtgttcaaa gtcaataatc aggtggtctc 1860

tttgaagcct gagattatcg tggatcaata tgagtacaag taccctgcca tcaaggattt 1920

gaaaaagcct tgtatcaccc tagggaaagc ccccgacttg aacaaagcat acaaatcagt 1980

tttatcaggc atgaatgccg ccaaacttga tccggatgat gtatgctcct acttggcagc 2040

agcaatgcag ttctttgagg ggacatgtcc ggaagactgg accagctatg gaatcctgat 2100

tgcacgaaaa ggagatagga tcaccccaaa ctctctagtg gagataaagc gtactgatgt 2160

agaagggaat tgggctctga caggaggcat ggaattgaca agggacccca ctgtctctga 2220

acatgcatct ttagtcggtc ttctcctgag tctgtacagg ttgagcaaaa tatcaggaca 2280

gaacactggt aactataaga caaacattgc agataggata gagcagattt tcgagacagc 2340

accttttgtt aagatcgtgg aacaccatac cctaatgaca actcacaaga tgtgtgctaa 2400

ttggagtact ataccgaact tcagattttt ggccggaacc tacgacatgt ttttctcacg 2460

gattgagcat ctgtattcgg caatcagagt gggcacagtc gtcaccgctt atgaagactg 2520

ctcaggactg gtatcgttta cagggttcat aaagcagatc aatctcaccg caagggaagc 2580

aatactatat ttcttccaca agaactttga ggaagagata agaagaatgt tcgagccagg 2640

gcaagagaca gctgttcctc actcttattt catccacttc cgttcactag gcttgagtgg 2700

gaagtctcct tattcatcga atgctgtcgg tcatgtgttc aatctcattc actttgttgg 2760

atgctacatg ggtcaagtca gatctctaaa tgcgacggtt attgctgcat gtgcccctca 2820

tgagatgtct gttctagggg gctatttggg agaggaattc ttcggaaaag ggacatttga 2880

aagaaggttc ttcagagacg agaaagaact tcaagaatat gaggcggctg aactaacaaa 2940

gtccgacgtg gcactggcgg atgacggaac cgtcaactct gatgacgagg actatttctc 3000

tggtgaaacc agaagtccag aagctgtcta tactcgaatc atgatgaatg gaggtcgact 3060

gaagagatct catatacgga gatatgtctc agtcagttcc aatcatcaag cccgtccaaa 3120

ctcattcgcc gaatttttaa acaagacgta ttcgaatgac tcataatcta ga 3172

<210>6

<211>226

<212>DNA

<213>Cricket Paralysis Virus

<400>6

tctagaaaag caaaaatgtg atcttgcttg taaatacaat tttgagaggt taataaatta 60

caagtagtgc tatttttgta tttaggttag ctatttagct ttacgttcca ggatgcctag 120

tggcagcccc acaatatcca ggaagccctc tctgcggttt ttcagattag gtagtcgaaa 180

aacctaagaa atttacctgc tacatttcaa gataccatgg agatct 226

Claims (12)

1, a kind of method for preparing rabies virus antigen comprises: the antigen gene of rabies virus or the expression combination of uniting of antigen gene are cloned into respectively in the baculovirus delivery carrier, make up and obtain shifting expression vector; Structure is obtained shifting expression vector and baculovirus DNA carry out cotransfection so that homologous recombination or swivel base to take place, transfer to respectively on the genome of baculovirus and obtain recombinant baculovirus expressing combination uniting of the antigen gene of rabies virus or antigen gene; With recombinate shape virus infection insect host and cell; Cultivate infected insect host and express corresponding rabies antigen; Results and the expressed antigen of purifying, promptly.

2, in accordance with the method for claim 1, it is characterized in that: the antigen gene of described rabies virus is rabies virus antigen protein G gene or rabies virus antigen protein N gene; The expression of uniting of the antigen gene of described rabies virus is combined as the nucleotide sequence shown in the SEQ ID NO:5.

3, according to the method for claim 1, it is characterized in that: the expression cassette of described baculovirus delivery carrier is combined by polyhedrin promotor, p10 promotor or ie-1 promotor and enhanser.

4, method according to claim 1 is characterized in that: described baculovirus delivery carrier is selected from pBM034, pBM93, AcRP23-lacZ, AcRP6-SC, AcUW1-lacZ, BacPAK6, Bac to Pac, Bacmid, BlucBacII (pETL), p2Bac, p2Blue, p89B310, pAc360, pAc373, pAcAB3, pAcAB 4, PAcAS3, pAcC129, pAcC4, DZI, pAcGP67, pAcIE1, pAcJP1, pAcMLF2, pAcMLF 7, pAcMLF8, pAcMP1, pAcMP2, pAcRP23, pAcRP25, pAcRW4, pAcsMAG, pAcUW1, pAcUW21, pAcUW2A, pAcUW2B, pAcUW3, pAcUW31, pAcUW41, pAcUW42, pAcUW43, pAcUW51, pAcVC2, pAcVC3, pAcYM1, pAcJcC5, pBac1, pBac2, pBlueBacIII, pBlueBacHis, pEV55, mXIV, pIEINeo, pJVETL, pJVNhel, pJVP10, pJVrsMAG, pMBac, pP10, pPAK1, pPBac, pSHONEX 1.1, pSYN XIV VI+, pSYNVI+wp, pSYNXIV VI-, pVL1391, pVL 1392, pVL1393, pVL941, pVL 945, pVL 985, pVTBac, pBM030 or pUAC-5.

5, according to the method for claim 1, it is characterized in that: constructed transfer expression vector is pVL1393 (G), pVL1393 (N) or pVL1393 (G-IRES-N); Perhaps constructed transfer expression vector is to carry out rabies virus antigen protein G gene and N expression of gene simultaneously with the IRES sequence in insect.

6, according to the method for claim 1, it is characterized in that: described baculovirus DNA is selected from BmNPV, AcMNPV, ApNPV, HaNPV, HzNPV, LdMNPV, MbMNPV, OpMNPV, S1MNPV, SeMNPV or SpltNPV.

7, according to the method for claim 6, it is characterized in that: described BmNPV is Bm-NPV-ZJ8.

8, according to the method for claim 1, it is characterized in that: described recombinant baculovirus is selected from following any one recombinant virus: (1) recombinant bombyx mori nuclear polyhedrosis virus rBmNPV (G); (2) recombinant bombyx mori nuclear polyhedrosis virus rBmNPV (N); (3) recombinant bombyx mori nuclear polyhedrosis virus rBmNPV (G-IRES-N).

9, method according to claim 1 is characterized in that: described insect host comprises silkworm (Bombyx mori), wild silkworm (Bombyx mandarina), Semen Ricini silkworm (Philosamia cynthia ricim), wild silkworm (Dictyoplocajapanica), Philosamia cynthia (Philosamia cynthia pryeri), tussah (Antheraea pernyi), yamama (Antheraea yamamai), wild giant silkworm (Antheraea polyphymus), autographa california (Atographa califorica), tea geometrid (Ectropis obliqua), cabbage army worm (Mamestra brassicae), prodenia litura (Spodoptera littoralis), autumn mythimna separata (Spodopterafrugiperda), cabbage looper (Trichoplusia ni), armyworm (Thaumetopoea wilkinsoni), bollworm (Heliothisarmigera), U.S. bollworm (Heliothis zea), oriental tobacco budworm (Heliothis assulta), cigarette beetle (Heliothisvirescens), oriental armyworm (Pseudaletia separata) or gypsymoth (Lymantria dispar) and corresponding insect cell.

10, according to the method for claim 1, it is characterized in that: described infection is meant that recombinant baculovirus infects the 1-5 insect larvae or the pupal cell in age by eating or seeing through epidermis, be preferably:, after infecting 3-6 days, collect body fluid or the tissue homogenate that contains antigenic silkworm larva of various rabies or pupa the silkworm larva or the pupa in recombinant silkworm baculovirus infection bombyx mori cell or percutaneous puncture-inoculation 1-5 age; Described pupal cell is 1-2 days an early stage tender pupa.

11, the rabies virus antigen for preparing by any one described method of claim 1-10.

12, a kind ofly prevent or treat rabic injection or oral vaccine, it is characterized in that: go up the described rabies virus antigen of claim 11 of significant quantity and the lipid acid that concentration is 25-35% forms through ultrasonic emulsification by treatment; Preferably, described lipid acid is made up of each component of following volume percent: palmitinic acid 7%, oleic acid 20.5%, 3% stearic acid, surplus is a water.