CN102304529B - Preparation method of rabbit hemorrhagic fever virus empty capsid antigen - Google Patents

Preparation method of rabbit hemorrhagic fever virus empty capsid antigen Download PDF

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CN102304529B
CN102304529B CN 201110254439 CN201110254439A CN102304529B CN 102304529 B CN102304529 B CN 102304529B CN 201110254439 CN201110254439 CN 201110254439 CN 201110254439 A CN201110254439 A CN 201110254439A CN 102304529 B CN102304529 B CN 102304529B
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hemorrhagic fever
fever virus
rabbit hemorrhagic
gene
recombinant
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CN102304529A (en
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张志芳
李轶女
易咏竹
王树坤
石小峰
王石宝
张渭蛟
舒惠国
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Biotechnology Research Institute of CAAS
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Abstract

The invention relates to the field of genetic engineering and particularly relates to a preparation method of a rabbit hemorrhagic fever virus empty capsid antigen. The method provided by the invention comprises the following steps: 1) constructing a rhabdovirus transfer carrier containing a rabbit hemorrhagic fever virus capsid protein VP60 gene or an optimized gene, wherein codon optimization is performed according to the codon frequency of bombyx mori; 2) performing cotransfection on the constructed transfer expression carrier and DNA (deoxyribonucleic acid) of rhabdovirus so as to carry out homologous recombination or transposition to further obtain the recombinant rhabdovirus; 3) infecting the recombinant rhabdovirus with the host cells of an insect; and 4) culturing the infected host of the insect to express the corresponding rabbit hemorrhagic fever virus empty capsid antigen, and harvesting and purifying the expressed antigen. By adopting the method provided by the invention, the production cost of the rabbit hemorrhagic fever virus empty capsid antigen can be greatly reduced, and the method has a plurality of advantages of safety, high efficiency, low energy consumption, low cost and the like.

Description

A kind of preparation method of rabbit hemorrhagic fever virus empty capsid antigen
Technical field
The present invention relates to the genetically engineered field, relate in particular to a kind of preparation method of rabbit hemorrhagic fever virus empty capsid antigen.
Background technology
Rabbit hemorrhagic disease is commonly called as the rabbit pest, by rabbit hemorrhagic disease virus (Rabbit hemorrhagic disease virus, RHDV) a kind of acute, strong, height contact, the lethality transmissible disease that cause, once be a kind of crushing transmissible disease of rabbit, because bring huge financial loss to rabbit keeping, enjoyed the concern of rabbit keeping.Within 1984, first Chinese has been reported this disease.1989, OIE (OIE) formally classified this disease as the category-B transmissible disease, and China classifies them as two class transmissible diseases (Wang Yongkun etc., the diagnosis and treatment of rabbit pest, 1992).
In (ICTV) the 7th time report of ICTV in 2000, listed the rabbit hemorrhagic fever virus in Caliciviridae (Caliciviridae) Lagovirus (Lagovirus).The RHDV virus particle is spherical in shape, without cyst membrane, and diameter 32-36nm, 20 body symmetries (Zhang Lihong etc., Guangdong animal and veterinary science and technology, 31:9-11,2006), its capsid consists of the cylindric capsomere of 32 high 5-6nm, the multiple copy of major structural protein VP60, is consisted of.Negative staining Electronic Speculum surface has the typical cup-shaped morphological structure of calicivirus, is also shown in the virus hollow capsid that minority does not have core under Electronic Speculum.
The RHDV genome is containing 2 open reading frame, the long open reading frame of 5 ' end (ORF1) the 2344 amino acid whose polyprotein precursors of encoding, it further is decomposed into capsid protein and a plurality of Nonstructural Protein by virus protease, wherein capsid protein is viral major structural protein, be called VP60, directly related with the immune response that the inducing anti-disease poison infects.The short open reading frame frame (ORF2) of 3 ' end, between 7025-7378bp, another little constituent of coding virus, be called VP10.
The N-end of VP60 forms the inner area of capsid protein, and the C-end forms the outside of capsid protein.Amino acid between the 31-250 of N-end is main anti-infectious immunity determining area.Research to VP60 albumen is found, during this capsid protein of vivoexpression, do not having under the condition of other any compositions, can naturally aggregate into do not wrap up nucleic acid, with natural RHDV virus particle similar virus-like particle (Rob N on physical aspect, Virus-like particle as immunogens, Trends in Microbiology, 11:438-444,2003).
In recent years, the application virus-like particle more and more is subject to numerous scientists' attention as the antigen vectors of vaccine.Virus-like particle is the particle form characteristics of simulated virus, the chimeric particle that the exogenous genetic fragment product is presented to the virion surface and forms, its formalness is identical with virion, even also has the native ligand of some virus receptor, simultaneously, also specific antigens of other cause of diseases etc. can be presented to the surface at oneself, thereby play an important role in the vaccine research of pathogenic agent.VLPs often can induce and produce than inactivated vaccine and the more strong humoral immunoresponse(HI) of soluble polypeptide; because glycoprotein antigen is presented with noninfective graininess simulation natural antigen presentation in its surface; and the immunne response that this method offers to cause is better than dissolved state; therefore the immunne response that the glycoprotein antigen played an important role in provide protection causes is expected more to approach natural infection; like this, virus-like particle more likely is widely used in the development of vaccine.And VLPs is owing to not wrapping up nucleic acid, not reproducible, therefore also there is no infectivity, is a kind of safe antigen vectors (Nagesha H S, Virus-like particles of calicivirus as epitope carriers, Arch Virol, 144:2429-2439,1999).
A large amount of experiments show, RHDV can not breed on the chicken embryo, also are difficult to stable propagation in various primary or passage cells.Now widely used vaccine is tissue inactivation seedling.Recently, the research of new generation vaccine mainly concentrates in the development of gene engineering vaccine, in the multiple expression systems such as intestinal bacteria, yeast saccharomyces cerevisiae, vaccinia virus and plant, has expressed VP60 albumen (Liu Huairan etc., animal medicine progress, 24:7-9,2003).Boga etc. at expression in escherichia coli the main capsid protein VP60 of RHDV Spain AST/89 strain, find, only with natural VP60, to have the part same antigen and not induce the generation protective immunity during with the beta-galactosidase enzymes expressing fusion protein as VP60; Can express the protein very similar to natural VP60 antigenicity and can induce in basic expression system and produce effective protective immunity take t7 rna polymerase, can resist the lethal hit of the RHDV that firmly purifies; Boga etc. express VP60 and can form virus-like particle in yeast saccharomyces cerevisiae; Famos etc. carry out high efficient expression by the VP60 of Spanish isolate AST/89 in yeast, and expressing quantity has carried out glycosylation modified up to 1.5g/L and about 70% expressing protein; Yan Weiwei etc. insert pPICZ B by RHDV VP60 gene to have the blood clotting characteristic and can be suppressed by the hyper-immune serum of anti-RHDV (Yan Weiwei etc., Chinese animal doctor's journal, 23:447-449,2003) through the recombinant protein of Pichia anomala expression.The carrier successful expression that Fernandez-Femandez etc. adopt plum pox potyvirus to build VP60, this expression product inoculation rabbit can be resisted attack (the Fernandez-Fernandez M R of lethal dose RHDV, Protection of rabbits against rabbit hemotthagic disease virus by immnization with the VP60 protein expressed in plan ts with a potyvirusbased vector, Virology, 280:283-291,2001); The extract that the employing potatos such as Castanon are expressed containing VP60 carries out immunity to rabbit; produce special antibody response and protection (the Castanon S to the RHDV strong virus attack can be provided; The effect ofthe promoter on expression of VP60gene from rabbit hemorrhagic disease virus in potato plants; Plant Science; 162:87-95,2002).
At present, still there are some problems in the research of RHDV recombinant vaccine: the VP60 of escherichia coli expression has insolubility and the relatively poor shortcoming of immune effect, and its application prospect is also pessimistic; Recombinant viral vaccine exists and spreads genetically modified organism safety problem to environment; Adopt the expression level of transgenic plant production VP60 at present also undesirable; Thereby development RHD is safe, new generation vaccine is imperative efficiently.
The rabbit pest are hemorrhage with respiratory system, organa parenchymatosum's oedema, extravasated blood and the hemorrhage feature that is changed to, and infected rabbits is everlasting dead in 48-72 hour.Can make tentative diagnosis according to lesion characteristic, the virus antigen detected in the rabbit tissue of dying of illness in conjunction with laboratory is made a definite diagnosis, and mainly uses blood coagulation tests (HA), enzyme linked immunosorbent assay (ELISA), immunoelectronmicroscopy and molecular biology method.
Insect bculovrirus expression vector system (BEVS) is since nineteen eighty-three sets up (Smith, Mol.Cell Biol., 3:2156-2165,1983), and existing nearly thousand kinds of foreign genes are expressed in this system.Its advantage is: the A:BEVS expression efficiency is high, and the biological activity of expression product is high, and its antigenicity, immunogenicity are all similar to native protein; B: silkworm baculovirus can hold larger foreign gene and not affect itself propagation; C: application polyhedrin gene promoter expression alien gene in late period in evening, even the recombination product is toxic to cell, does not affect expression level yet; D: borrow polynary expression vector or borrow several different recombinant virus coinfections, can express 2 or more foreign protein simultaneously, structure and the function of the supramolecule assembling of research peptide chain and albumen oligomer; E: baculovirus is to vertebrates without pathogenicity bo, and silkworm does not have the disease with infecting both domestic animals and human, and being considered on genetics is safe expression vector (Jing Zhizhong etc., Chinese animal doctor's science and technology, 31:43-45,2001).
Silkworm BmNPV expression system (silkworm biological reactor) has been widely used since exploitation in 1984 on medical science, veterinary science and agricultural.1997, it was carrier that the BmNPV modified is take in Hubei Province, field etc., in bombyx mori cell and larva successful expression schistosoma japonicum Chinese strain 28KD glutathione S-transferase gene (Acta Biochimica et Biophysica Sinica, 1997,29 (1): 33-38).Zhang Chuanxi etc. have obtained expression to the EPO gene in silkworm cultured cell and larva, and expression product has good external activity.Nineteen ninety, Chu Ruiyin etc. have obtained the HBsAg gene respectively expression in silkworm cultured cell and nutrient solution thereof.Zhou Naiming etc. in silkworm larva and pupa successful expression HBsAg, average every the silkworm of its output is about 750 μ g, each pupa is about 690 μ g, product mainly exists with the glycosylation form.The tens of kinds of animal virus antigen proteins such as foot and mouth disease virus successively are expressed and purifying, and wherein quite a few is carrying out commercialized development (Li Zhiyong etc., Plos one, e2273,2008).The prevention restructuring IL preparation (Intercat) of the pet (cat and dog) produced by the BmNPV system (the Kimura's taste of by Japan " TORAY " Co., Ltd., having been produced and put goods on the market, insect biological factory [M], Japan: census of manufacturing meeting, 2000,124-127).
The relevant technologies of baculovirus expression vector system is relatively ripe now, and this system expression foreign gene is not only economical, efficient, and a new technological approaches can be provided.Utilize silkworm biological reactor to produce the rabbit hemorrhagic fever virus empty capsid antigen, guarantee that it has normal protein structure and biology immunocompetence, for the commercial production of rabbit hemorrhagic fever vaccine lays the foundation.
Summary of the invention
The present inventor proposes and has completed the present invention in order to address the above problem.
The objective of the invention is to overcome the deficiencies in the prior art, provide utilize baculovirus expression vector system in insect body safety, express efficiently the method for rabbit hemorrhagic fever virus empty capsid antigen.
Another object of the present invention is to provide rabbit hemorrhagic fever virus capsid protein VP60 optimized gene.
A further object of the present invention is to provide the baculovirus transfer vector that comprises rabbit hemorrhagic fever virus capsid protein VP60 or above-mentioned optimized gene, and recombinant baculovirus.
A further object of the present invention is to provide the insect bio-reactor of preparation rabbit hemorrhagic fever virus empty capsid antigen.
A further object of the present invention is to provide the rabbit hemorrhagic fever virus empty capsid antigen prepared by aforesaid method.
A further object of the present invention is to provide the application as the rabbit hemorrhagic fever vaccine of the rabbit hemorrhagic fever virus empty capsid antigen for preparing by aforesaid method.
According to rabbit hemorrhagic fever virus capsid protein VP60 optimized gene of the present invention, its nucleotide sequence is as shown in SEQ ID No.2.
Further, the invention provides the baculovirus transfer vector that comprises rabbit hemorrhagic fever virus capsid protein VP60 or above-mentioned optimized gene.
The present invention also provides by using above-mentioned baculovirus transfer vector to infect the recombinant baculovirus that baculovirus obtains.
The present invention also provides the insect bio-reactor of preparation rabbit hemorrhagic fever virus empty capsid antigen, by using above-mentioned recombinate shape virus infection insect host, obtains.
Therefore, according to the preparation method of rabbit hemorrhagic fever virus empty capsid antigen of the present invention, comprise the following steps:
1) building the baculovirus transfer vector comprise rabbit hemorrhagic fever virus capsid protein VP60 gene or above-mentioned optimized gene, wherein, is that the codon frequency by silkworm carries out codon optimized;
2) the transfer expression vector and the baculovirus DNA that structure are obtained carry out cotransfection, so that homologous recombination or swivel base to occur, obtain recombinant baculovirus;
3) by the recombinate shape virus infection insect host cell;
4) cultivate infected insect host, express corresponding rabbit hemorrhagic fever virus empty capsid antigen, results the expressed antigen of purifying.
Preparation method according to rabbit hemorrhagic fever virus empty capsid antigen of the present invention, wherein, the original series of described rabbit hemorrhagic fever virus capsid protein VP60 gene and through the nucleotide sequence after codon optimized as shown in SEQ ID NO.1 and SEQ ID NO.2, the aminoacid sequence SEQ ID NO.3 of the viral VP60 of its coding.
According to the preparation method of rabbit hemorrhagic fever virus empty capsid antigen of the present invention, comprise, wherein, described baculovirus transfer vector is preferably from AcRP23-lacZ, AcRP6-SC, AcUW1-lacZ, BacPAK6, Bac to Pac, Bacmid, p2Bac, p2Blue, BlucBacH (pETL), p89B310, pAc360, pAc373, pAcAB3, pAcAB 4, PAcAS3, pAcC 129, 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, pJVNhe1, pJVP10, pJVrsMAG, pMBac, pP10, pPAK1, pPBac, pSHONEX 1.1, pSYN XIV VI+, pSYNVI+wp, pSYNXIV VI-, pVL1391, pVL 1392, pVL 1393, pVL941, pVL 945, pVL 985, pVTBac, pBM030, or pUAC-5, or other similar baculovirus homologous recombination or transposon vector, more preferably pVL1393 baculovirus delivery carrier.
Preparation method according to rabbit hemorrhagic fever virus empty capsid antigen of the present invention, wherein, constructed transfer vector is with the carrier pVL1393-RHDV-VP60 of rabbit hemorrhagic fever virus capsid protein VP60 original series with the carrier pVL 1393-RHDV-VP60-O of rabbit hemorrhagic fever virus capsid protein VP60 gene sequence after codon optimized.
Preparation method according to rabbit hemorrhagic fever virus empty capsid antigen of the present invention, wherein, described baculovirus preferably from BmNPV, AcMNPV, ApNPV,, HaNPV, HzNPV, LdMNPV, MbMNPV, OpMNPV, SlMNPV, SeMNPV or SpltNPV, more preferably silkworm baculovirus parent plant Bm-NPV-ZJ8.
According to the preferred embodiment of the present invention, wherein, described recombinant baculovirus is selected from following any one recombinant virus: (1) recombinant bombyx mori nuclear polyhedrosis virus rBmNPV (RHDV-VP60), (2) recombinant bombyx mori nuclear 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 lepidopterous insects, as silkworm (Bombyx mori), wild silkworm (Bombyx mandarina), Semen Ricini silkworm (Philosamia cynthia ricim), wild silkworm (Dictyoplca japanica), 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 (Spodoptera frugiperda), cabbage looper (Trichoplusia ni), armyworm (Thaumetopoea wilkinsoni), bollworm (Heliothis armigera), U.S. bollworm (Heliothis zea), oriental tobacco budworm (Heliothis assulta), cigarette beetle (Heliothis virescens), oriental armyworm (Pseudaletia separata), gypsymoth (Lymantria dispar) etc., silkworm (Bombyx mori) more preferably.
Preparation method according to rabbit hemorrhagic fever virus empty capsid antigen of the present invention, wherein, described infection refers to that recombinant baculovirus infects the insect larvae in 1-5 age or pupal cell (more preferably: by silkworm larva or the pupa in recombinant Bombyx mori baculovirus infected silkworm cell or percutaneous puncture-inoculation 1-5 age, within 3-6 days, collect afterwards containing the silkworm larva of rabbit hemorrhagic fever virus empty capsid antigen or body fluid or the tissue homogenate of pupa infecting) by eating or seeing through epidermis; Wherein, the early stage tender pupa that described pupal cell optimum is 1-2 days.
The present invention adopts gene recombination technology, the capsid protein gene sequence of rabbit hemorrhagic fever virus (RHDV) is carried out codon optimized, by the original series of this albumen (SEQ ID NO:1) and the sequence (SEQ ID NO:2) after optimizing be cloned into baculovirus transfer vector (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+wp, pSYNXIV VI-, pVL1391, 1392, 1393, pVL941, 945, 985, pVTBac, pBM030, pUAC-5) upper, in the polyhedron promotor, under p10 promotor or other virus and Eukaryotic strong promoter are controlled, by in body or external (in vivo/in vitro) restructuring, the original series of RHDV capsid protein and the sequence after optimization are incorporated on the genome of baculovirus, obtain recombinant virus, recombinant virus can be by adding food or adopting various means to see through insect larvae or pupal cell (the early stage tender pupa that optimal time is 1-2 days) that epidermis infects 1-5 age (optimal time was four or five ages), Expression product rabbit hemorrhagic fever virus empty capsid antigen.
According to the preferred embodiment of the present invention, capsid protein gene sequence to rabbit hemorrhagic fever virus (RHDV) is carried out codon optimized, by the sequence after the original series of this capsid protein and optimization, be that the base sequence shown in SEQ ID NO.1 and SEQ ID NO.2 is cloned on baculovirus delivery carrier pVL1393, again by body the restructuring by the original series VP60 of rabbit hemorrhagic fever virus capsid protein gene and codon optimized after sequence VP60-O transfer to respectively on the genome of silkworm baculovirus parent plant Bm-NPV-ZJ8, substitute the Polyhedrin gene on genome, by plaque select technology and PCR detection technique, the recombinant Bombyx mori baculovirus rBmNPV (HDV-VP60) of rabbit hemorrhagic fever virus capsid protein gene is carried in acquisition, rBmNPV (HDV-VP60-O), by silkworm larva or the pupa in its infected silkworm clone or percutaneous puncture-inoculation 1-5 age, amount reproduction rBmNPV (HDV-VP60), rBmNPV (RHDV-VP60-O), when rBmNPV (RHDV-VP60), rBmNPV (RHDV-VP60-O) copy in the silkworm body, VP60 and VP60-O gene are expressed under polyhedron gene (polh) promotor is controlled, and self assembly becomes the rabbit hemorrhagic fever virus empty capsid antigen, infecting, within 3-6 days, (the best is 5 days, 25 ℃ of raising temperatures) after, collect containing the silkworm larva of rabbit hemorrhagic fever virus empty capsid antigen or the body fluid (or whole homogenate) of pupa, just obtain safety, efficient rabbit hemorrhagic fever virus empty capsid antigen after radiation irradiation is killed baculovirus, protein purification, this antigen can be used for the injection vaccine of preparation prevention rabbit hemorrhagic disease.
The inventive method adopts baculovirus expression system to produce safely, efficiently rabbit hemorrhagic fever virus antigen in silkworm biological reactor, its production cost is significantly lower than traditional method for preparing rabbit hemorrhagic fever virus antigen, found the factory, the three wastes, electric power and the consumption of water resources equal energy source are few.Because silkworm is approved as food medicine dual-purpose insect by China Ministry of Health, so by the inventive method after prepared antigen purification, security is high, can directly make the vaccine immunity animal.
In general, the production cost that the inventive method can decrease rabbit hemorrhagic fever virus empty capsid antigen, have the plurality of advantages such as safety, efficient, less energy consumption, cost are low.
The accompanying drawing explanation
The electron-microscope scanning figure of the RHDV hollow capsid particle that Fig. 1 is silkworm production;
Fig. 2 is the hollow capsid immuno-gold labeling electron-microscope scanning figure that silkworm produces.
Embodiment
Produce in detail the present invention below in conjunction with embodiment and accompanying drawing, those having ordinary skill in the art will appreciate that these examples are illustrative fully, do not limit the scope of the invention.
Experiment material
E. coli strains E.coli DH 5 αpurchased from Promega company; Cloning vector pEASY-T3 is purchased from full formula King Company; Cloning vector pMD18-T is purchased from TaKaRa company; Prokaryotic expression carrier pET-28a +, recipient bacterium E.coli TOP10 and BL21 (DE3) be conventional bacterial strain purchased from Novagen company, delivery carrier pVL1393 is purchased from Invitrogen company; The original series VP60 of rabbit hemorrhagic fever virus capsid protein gene extracts and is cloned into carrier to obtain from morbidity animal pathological tissues, and the sequence VP60-O direct gene after codon optimized is synthesized and obtained; Bombyx mori cell BmN, Bombyx mori nuclear polyhydrosis virus parent plant Bm-NPV-ZJ8 are so kind as to give by the school of life and health sciences Wu Xiangfu researcher of the Chinese Academy of Sciences, and molecule Microbiological Lab of Biological Technology institute, Chinese Academy of Agricultural Sciences preserves; High expression level cultivated silkworm breed variety JY1 is by the seed selection of silkworm industry Science Institute of the Chinese Academy of Agricultural Sciences, and Biological Technology institute, Chinese Academy of Agricultural Sciences preserves.
Enzyme and reagent: restriction enzyme used and supporting damping fluid are all purchased from Promega company; T4 DNA Ligase and damping fluid are Promega company product; LA Taq polysaccharase and damping fluid are purchased from TaKaRa company; RnaseA, dNTPs are purchased from Sigma company; The DNA of all size and protein molecular weight standard are TranGen Biotech company product; DEPC, M-MLV-Rtase (reversed transcriptive enzyme) are purchased from Promega company.
Biochemical reagents: bisacrylamide, acrylamide, IPTG, X-Gal are purchased from Promega company; Tris, Ampicillin, Kanamycin, IPTG, SDS, urea, imidazoles, TritonX-100, TEMED (N, N, N ', N '-Tetramethylethylene diamine), ammonium persulphate (Ammonium Persulfate), kantlex (Kanamycin), cell culture medium TC-100 are purchased from Sigma company; Agarose is Sunbiotech company product; Yeast extract (Yeast Extract), Tryptones are all purchased from Britain OXOID company; 0.2um, the 0.45um filter is purchased from Gelman Sciences company; Ethidium bromide (EB), Coomassie brilliant blue R-250 are purchased from Fluka company; Agar powder is Japanese import packing; Ni-NTA resin, Proteinase K, foetal calf serum are purchased from Invitrogen company; Other is domestic or Import Analysis pure reagent.It is synthetic that primer is won polygala root biotechnology limited liability company by three
Substratum: Escherichia coli culture medium is the LB substratum; The bombyx mori cell substratum is the TC-100 substratum.
The experimentation on animals of rabbit hemorrhagic fever virus capsid protein gene expression product is carried out at isolation laboratory.
The solubility expression of original series VP60 in silkworm biological reactor and the detection of expression product of embodiment 1, rabbit hemorrhagic fever virus capsid protein gene
1, the acquisition of the original series VP60 of rabbit hemorrhagic fever virus capsid protein gene
The TRIzol method is extracted rabbit illing tissue geneome RNA.
Design primer, amplify the original series VP60 (SEQ ID NO.1) of rabbit hemorrhagic fever virus capsid protein gene by the method for RT-PCR.Designed reverse transcription special primer is: RHDV-RT:5 '-GGATTAAAACCTAACCTACC-3 '.The amplimer of the original series VP60 of capsid protein gene is: the VP60 upstream: 5 '-G gAATTCaACATGGAGGGCAAAGCCCGCACAG-3 ' (EcoR I); The VP60 downstream: 5 '-GA aGATCTtCAGACATAAGAAAAGCCATTG-3 ' (Bgl II).
Pcr amplification is analyzed clip size by agarose gel electrophoresis after finishing.Reclaim the goal gene fragment, be connected (pMD18-T) with cloning vector and connect.Transformed E .coli heat shock competent cell, identify positive recombinant plasmid.The bacterial classification that will be accredited as positive colony (called after pT-RHDV-VP60) adds the LB nutrient solution that contains Amp again, 220r/min shakes training and spends the night rear, draw the fresh bacterium liquid of 1mL to aseptic Eppendorf pipe, add a small amount of glycerine, after sealing with sealed membrane, to Beijing, order-checking section of San Bo Bioisystech Co., Ltd is checked order, and result is as shown in SEQ ID NO.1, and aminoacid sequence is as shown in SEQ ID NO.3.For sequencing result, DNAStar, DNAMAN software are analyzed sequence.
2, the structure of baculovirus transfer vector pVL1393-RHDV-VP60
The recombinant plasmid pT-RHDV-VP60 that order-checking was identified cuts with EcoR I enzyme, makes its linearizing.Eukaryotic expression plasmid pVL1393 makes same enzyme and cuts processing.The RHDV-VP60 purpose fragment that enzyme cuts back to close is connected with transfer vector pVL1393's after EcoR I/Bgl II double digestion enzyme is cut.Recombinant plasmid is identified and gene sequencing through EcoR I/Bgl II double digestion, bacterium liquid PCR, is identified correct recombinant plasmid called after pVL1393-RHDV-VP60.
3, the preparation of the breeding of Bombyx mori nuclear polyhydrosis virus parent plant Bm-NPV-ZJ8 and viral DNA
Press Sigma company description of product preparation substratum, cultivate bombyx mori cell BmN under 27 ℃.Infect the about 50ml of cell of logarithmic phase with Bombyx mori nuclear polyhydrosis virus parent plant Bm-NPV-ZJ8, infection multiplicity is to collect virus infection liquid after 1,3~4 days, centrifugal (10000rpm * 10min), remove precipitation, supernatant is used centrifugal 1 hour of 25000rpm, except supernatant, (contain Tris 12.1g in 1000ml with 1ml viral DNA extract, EDTA 33.6g, KCl 14.1g, pH7.5) suspension virus particle precipitation, extracting DNA, put 4 ℃ and save backup.
4, structure and the acquisition of recombinant Bombyx mori baculovirus rBmNPV (RHDV-VP60)
Inoculate about 1 * 10 6cell, in the 15cm2 culturing bottle, after cell attachment, is removed containing foetal calf serum (FBS) substratum, with the substratum that does not contain FBS, washes three times, adds 1.5ml without the FBS substratum.Add successively 1 μ g silkworm baculovirus parent plant Bm-NPV-ZJ8DNA in a sterile tube, 2 μ g recombinant transfer plasmid pVL1393-RHDV-VP60DNA and 5 μ l liposomes, supply volume to 60 μ l with aseptic double-distilled water, mix gently, after standing 15min, dropwise join in culturing bottle and carry out cotransfection.Microscopic examination, will not contain polyhedrosis plaque and pick out, and repeat above step, through 2~3 purifying of taking turns, obtain pure recombinant Bombyx mori baculovirus rBmNPV (RHDV-VP60).
Recombinant Bombyx mori baculovirus rBmNPV (RHDV-VP60) is infected to the BmN cell of normal growth, cultivate after 3 days and collect supernatant liquor, contain a large amount of recombinant virus rBmNPV (RHDV-VP60) in supernatant.
Utilize PCR method to analyze exogenous origin gene integrator, Oligonucleolide primers is:
VP60 upstream: 5 ' G gAATTCaACATGGAGGGCAAAGCCCGCACAG-3 ' (EcoR I); The VP60 downstream: 5 '-GA aGATCTtCAGACATAAGAAAAGCCATTG-3 ' (Bgl II).
The result proof has obtained recombinant virus.
6, RHDV-VP60 high efficient expression in silkworm pupa and silkworm body
Silkworm pupa used is that the high expression level kind is JY1.JY1 kind silkworm rearing is pressed the ordinary method of " Chinese sericulture " (Shanghai science tech publishing house, 1991) of Lv Hongsheng chief editor and is carried out.After first feeding 48h select the silkworm that mean body weight is identical and cocoon seven days after 15 identical silkworm chrysalises of mean body weight, every silkworm chrysalis and silkworm inoculate approximately 1.0 * 10 5rBmNPV (RHDV-VP60), collect the morbidity silkworm chrysalis and get silkworm blood after 4-5 days ,-20 ℃ frozen to carry out the double antibody sandwich ELISA detection.
7, the collection of RHDV-VP60 virus-like particle and purifying.
Will be containing after expressing and having the silkworm chrysalis of RHDV-VP60 to grind in homogenizer with the PBS (solid-liquid ratio is 1: 9) of precooling, the filtration of use 0.45um filter.In 30% sucrose solution, 1.5 * 10 5centrifugal 2 hours of g ultra-high speed.With the solution of the Tris-HCl (PH7.0) containing 0.1M NaCl, precipitation is redissolved to original volume, cross cation-exchange chromatography filler SP (GE company), the Tris-HCl of 0.5M NaCl (PH7.0) wash-out.Again by sieve chromatography S200 (GE company).Purity 95%, yield can reach more than 40%.Prove, the target protein of expressing in silkworm can be self-assembled into the virus hollow capsid particle under high density, but also has set up the purification process of corresponding silkworm expression genetically engineered rabbit hemorrhagic disease virus empty capsid antigen simultaneously.
8, the detection of expression product
(1) ELISA and Western blotting detect
Silkworm after injecting virus is collected silkworm blood when falling ill.The ELISA method detects the height of RHDV-VP60 antigen protein expression amount, coating buffer is as blank, using normal silkworm blood as negative control, and the rabbit anteserum after the RHDV-FR2 protein immunization new zealand white rabbit of prokaryotic expression is first antibody, and the goat anti-rabbit antibody of HRP mark is second antibody.Appoint and get the sample in received silkworm blood, with coating buffer, do gradient dilution, from 50 * twice doubling dilution to 6400 times, do diplopore and detect at each gradient place, during calculating, averages, and respectively gets 100 μ L and be coated with on enzyme plate.Result, as table 1, shows that RHDV-VP60 gene expression amount in silkworm is high, and the expression amount in average every boss silkworm or silkworm chrysalis is 1mg at least, at 3200 times, dilutes even and still the specific reaction of antigen and antibody can be detected under greater dilution.
Table 1ELISA detects the VP-60 gene that silkworm biological reactor is expressed
Western blotting result shows can detect in the supernatant liquor of silkworm hemolymph sample after recombinant virus infection the specific band of 60kD size.
(2) blood coagulation tests of the rabbit hemorrhagic disease virus empty capsid antigen of expressing in silkworm detects
Get " O " type red corpuscle of people and preserve in A Shi liquid, with the physiological saline Washed Red Blood Cells of 20 times three times, finally red corpuscle is made into to 1% suspension with physiological saline.The physiological saline 50ul that adds sterilizing in every hole of U-shaped plate; Get adding in the 1st hole containing virus hollow capsid solution dilution liquid 50ul of suitable multiple dilution with micropipet, after mixing, join in the second hole after absorption 50ul, serial dilution to the like this 10 holes, the 10th hole is drawn 50ul liquid and is discarded; The 11st hole and the 12nd hole are the physiology saline control, and negative silkworm blood is by the same procedure dilution.Every hole adds 50ul1% people's " O " type red cell suspension, and after mixing, reaction is 45 minutes under 4 ℃, observations after the red corpuscle contrasted deposits fully.When the rabbit hemorrhagic disease virus empty capsid antigen of preliminary purification is diluted to 64000 times for still positive.
(3) animal immune experiment.
By the purifying antigen RHDV viral capsid particle of collection, through intramuscular injection and 10 rabbit of each immunity of oral route, be test group, what separately establish 5 rabbit injection adjuvants and feeding antigen is control group, and rabbit is the RHDV feminine gender.5ug RHDV empty capsid antigen mixes the trial-production vaccine with oily adjuvant equal-volume, 1ml/ only, in the enterprising action thing test of rabbit, gets blood after 21 days, carries out the detection of ELISA antibody titer, test group all produces the antibody for RHDV, and the injection group detects to tire and can reach more than 3200 times; Empty capsid particle by preliminary purification, amount by every rabbit 50 μ g is mixed with the propolis containing 25%-35% lipid acid and 8%-12% of same volume, optimum concn is lipid acid 30%+ propolis 10% (it is 3% that lipid acid consists of palmitinic acid 8%, oleic acid 19%, other composition), after ultrasonic emulsification, pour into, after one month, oral group of detection IgA tires and also reaches 1600 times.Test group 100% produces protection antibody as a result, and control group does not detect antibody; Attacked poison experiment when getting blood, though the injection of immune group or orally all there is no rabbit death, and control group is attacked all death in latter 3 days of poison, shows typical rabbit hemorrhagic fever symptom.
Embodiment 2, the solubility expression of the sequence VP60-O of rabbit hemorrhagic fever virus capsid protein gene after codon optimized in silkworm biological reactor and the detection of expression product
1, optimize the acquisition of rear sequence VP60-O
Rabbit hemorrhagic fever virus capsid protein gene original series VP60 embodiment 1 recorded according to the silkworm codon preference is optimized, do not change aminoacid sequence, after optimizing, sequence VP60-O is as shown in SEQ ID NO.2, the rare codon that contains series connection in original series, this has reduced translation sequences or has even removed translating equipment, after optimizing, sequence CAI value rises to 0.79 by 0.72, GC content and unsuitable peak have been adjusted to extend the transformation period of mRNA, GC content is adjusted into 53.34% by 54.9%, those affect mRNA stability and the loop-stem structure of being combined with rrna destroyed, Bam HI restriction enzyme site is arranged in 90 sites in original series, there is Xho I restriction enzyme site in 487 sites, sequence after optimization is without these two restriction enzyme sites.Directly synthesize the sequence VP60-O after optimizing and be cloned into carrier pUC57 above, two ends are respectively with Bam HI, EcoR I restriction enzyme site.Order-checking is identified by analyzing to obtain SEQ ID NO:2 and corresponding aminoacid sequence SEQ ID NO.3.
2, the structure of recombinant baculovirus transfer vector pVL1393-RHDV-VP60-O
The recombinant plasmid pUC57-RHDV-VP60-O that order-checking was identified cuts with Bam HI enzyme, makes its linearizing.Eukaryotic expression plasmid pVL1393 does same the processing.
Being connected through the product of Bam HI/EcoR I double digestion and transfer vector pVL1393 after Bam HI/EcoR I double digestion by plasmid pUC57-RHDV-VP60-O.
To connect product transformed competence colibacillus cell, identify correct recombinant plasmid called after pVL1393-RHDV-VP60-O.
3, the preparation of the breeding of Bombyx mori nuclear polyhydrosis virus parent plant Bm-NPV-ZJ8 and viral DNA
With embodiment 1.
4, structure and the acquisition of recombinant Bombyx mori baculovirus rBmNPV (RHDV-VP60-O)
With embodiment 1.
Recombinant Bombyx mori baculovirus rBmNPV (RHDV-VP60-O) is infected to the BmN cell of normal growth, cultivate after 3 days and collect supernatant liquor, contain a large amount of recombinant virus rBmNPV (RHDV-VP60-O) in supernatant liquor.Utilize PCR method to analyze exogenous origin gene integrator.Oligonucleolide primers is: by two primers of the VP60-O gene order indoor design of optimizing, identify sequence:
RHDV-F:5′-ACCTTGGTCCTTTCCGTATATAA-3′;
RHDV-R:5′-CTTGGCGACAGTTTGAGCAC-3′。
The result proof has obtained recombinant virus.
5, RHDV-VP60-O high efficient expression in silkworm pupa and silkworm body
Silkworm pupa used is that the high expression level kind is JY1.JY1 kind silkworm rearing is pressed the ordinary method of " Chinese sericulture " (Shanghai science tech publishing house, 1991) of Lv Hongsheng chief editor and is carried out.After first feeding 48h select the silkworm that mean body weight is identical and cocoon seven days after 15 identical silkworm chrysalises of mean body weight, every silkworm chrysalis and silkworm inoculate approximately 1.0 * 10 5rBmNPV (RHDV-VP60-O), collect the morbidity silkworm chrysalis and get silkworm blood after 4-5 days ,-20 ℃ frozen to carry out the double antibody sandwich ELISA detection.
6, the collection of RHDV-VP60-O virus-like particle and purifying
Will be containing after expressing and having the silkworm chrysalis of RHDV-VP60-O to grind in homogenizer with the PBS (solid-liquid ratio is 1: 9) of precooling, the filtration of use 0.45um filter.In 30% sucrose solution, 1.5 * 10 5centrifugal 2 hours of g ultra-high speed.With the solution of the Tris-HCl (PH7.0) containing 0.1MNaCl, precipitation is redissolved to original volume, cross cation-exchange chromatography filler SP (GE company), the Tris-HCl of 0.5M NaCl (PH7.0) wash-out.Again by sieve chromatography S200 (GE company).Purity 95%, yield can reach more than 40%.Prove, the target protein of expressing in silkworm is soluble, but also has set up the purification process of corresponding silkworm expression genetically engineered rabbit hemorrhagic disease virus empty capsid antigen simultaneously.
7, rabbit hemorrhagic disease virus detects the preparation of antibody
Design three pairs of special primers and divide three sections to carry out prokaryotic expression the capsid protein sequence VP60-O after optimizing, design of primers is 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 recombination plasmid pT3-RHDV-FR1, pT3-RHDV-FR2 and pT3-RHDV-FR3
Take plasmid pUC57-RHDV-VP60-O as template, use respectively above-mentioned three couples of primer amplification purpose fragment RHDV-FR1, RHDV-FR2, RHDV-FR3; Glass milk method purifying reclaims the PCR product, the PCR purified product is connected with cloning vector pEASY-T3, connect product and transform the intestinal bacteria thermal shock competent cell prepared, choose spot and cultivate the evaluation of carrying out recombinant plasmid, the bacterial strain that after the disrupt red cell Rapid identification, picking plasmid band is stepped back, alkaline lysis method of extracting recombinant plasmid pT3-RHDV-FR1, pT3-RHDV-FR2 and pT3-RHDV-FR3, carry out the double digestion evaluation with Bam HI/Hind III, and enzyme is cut and identified that correct recombinant bacterium bacterium liquid send three rich polygala root companies to carry out the DNA sequencing checking.Sequencing result is carried out to nucleotide sequence with the sequence of previous plasmid pUC57-RHDV-VP60-O and compare, result shows that three aim sequences in recombinant plasmid are consistent with previous plasmid sequence result, the restriction enzyme site that phase shift mutation does not occur and contain design.
(2) structure of recombinant plasmid pET28a-RHDV-FR1, pET28a-RHDV-FR2, pET28a-RHDV-FR3
Check order correct recombinant plasmid pT3-RHDV-FR1, pT3-RHDV-FR2 and pT3-RHDV-FR3 after the digestion of Bam HI/Hind III double digestion, glass milk method purifying reclaims enzyme and cuts product, be connected to by the expression vector pET-28a of Bam HI/Hind III double digestion digestion, connect product and transform the Top10 competent cell, first carry out the disrupt red cell Rapid identification after choosing spot, then a small amount of extracting recombinant plasmid pET28a-RHDV-FR1, pET28a-RHDV-FR2, pET28a-RHDV-FR3 fast of alkaline process, identify with Bam HI/Hind III double digestion.
(3) recombinant plasmid is induced amalgamation and expression in intestinal bacteria
Enzyme is cut correct above-mentioned recombinant plasmid and is transformed respectively the BL21 competent cell, IPTG collects bacterium liquid after inducing 4h, with SDS-PAGE electrophoretic analysis expression, result shows to be compared with negative control, and the expressing fusion protein band that transforms the bacterial strain appearance of recombinant plasmid pT3-RHDV-FR2 concentrates most.After the best expression strain list bacterium colony shaking culture of picking effect, induced, after ultrasonication, the fusion rotein (His-RHDV-FR2) of SDS-PAGE electrophoretic analysis Explicit Expression is present in precipitation, shows that expressing protein exists with the inclusion body form.
(4) preparation of the purifying of fusion rotein and antibody thereof
Carry out a large amount of abduction deliverings after the expression strain list bacterium colony shaking culture that picking is successfully recombinated, the related solution of employing processing inclusion body protein and method are by after solubilization of inclusion bodies, with this expressing protein of Ni+-NTA resin chromatography column purification, at urea NTA-25, urea NTA-50, urea NTA-100, urea NTA-250, urea NTA-500,5 gradients are collected elutriant, collection penetrates liquid, elutriant, every pipe is collected a NTA volume, the distribution situation in elutriant in conjunction with situation, target protein of SDS-PAGE Analysis deterrmination protein.Result shows the single band that size is correct, the target protein His-RHDV-FR2 size of expressing is consistent with positive control, while with urea NTA-100, carrying out wash-out, and the elution peak maximum, cut off purifying band albumen with the sterilizing pocket knife after SDS-PAGE glue purification albumen, and be cut into 1mm 3little blob of viscose, send Institute of Zoology, Academia Sinica's microbial film and film biotechnology National Key Laboratory to carry out mass spectroscopy, the database search identification method that method is second order ms, the show sample protein sequence detects fraction of coverage 34.82% as a result, analyze and show, this sample Score Delta Cn surpasses 30, have 2 sections peptide sections can deserve and wherein 1 section peptide section aminoacid sequence be greater than 50, the AA sequence that can detect and expected sequence matching rate are up to 34.68%, so can determine that purifying protein is exactly the expression product of target gene RHDV-FR2.The fusion rotein that purifying is expressed to carry out albumen concentrated, adopt the Bradford method survey concentrated after the concentration of protein solution.The concentration that purifying is good is greater than 1mg/ml, after the fusion rotein that total amount is not less than 3mg carries out SDS-PAGE, cuts off the purpose band, after micelle is ground, send in Chinese Academy of Sciences's heredity with grow institute, the negative rabbit of immune RHDV prepares polyclonal antibody.
(5) polyclonal antibody bioactivity
Obtain polyclonal antibody after the RHDV-FR2 protein immunization new zealand white rabbit of purifying 4 times, make envelope antigen with the fusion rotein of purifying, make primary antibodie with the polyclonal antiserum of PBS dilution different multiples 100,200,400,800,1600,3200,6400,12800,25600,51200, the sandwich ELISA detection method is surveyed antibody titer figure, before immunity, rabbit anteserum is made negative control, at A492nm place light absorption value, take each extension rate as X-axis, the light absorption value mean value under each extension rate is Y-axis.Usually be greater than 2.1 times of negative control OD value of regulation, i.e. positive (to calculate after the zeroing of blank hole), result shows: when homemade antibody is 10 μ g/ml in antigen protein concentration, tiring of antibody can reach 1: 3200.
8, the detection of expression product
(1) ELISA and Western blotting detect
Silkworm after injecting virus is collected silkworm blood when falling ill.The ELISA method detects the height of RHDV-VP60-O antigen protein expression amount, coating buffer is as blank, using normal silkworm blood as negative control, and the rabbit anteserum after the RHDV-FR2 protein immunization new zealand white rabbit of prokaryotic expression is first antibody, and the goat anti-rabbit antibody of HRP mark is second antibody.Appoint and get sample in received silkworm blood, do gradient dilution with coating buffer, from 100 * twice doubling dilution to 6400 times.Do diplopore and detect at each gradient place, respectively gets 100 μ L and be coated with on enzyme plate.Result is as table 2, show that RHDV-VP60-O gene expression amount in silkworm is high, exceed 2-3 doubly than RHDV-VP60 gene expression amount in silkworm, at 6400 times, dilute the specific reaction (as shown in table 2) that antigen and antibody still can be detected under greater dilution even.
Table 2ELISA detects the VP60-O gene of expressing in silkworm biological reactor
Western blotting result shows can detect in the supernatant liquor of hemolymph sample of silkworm after recombinant virus infection the specific band of 60kD size.
(2) blood coagulation tests of the rabbit hemorrhagic disease virus empty capsid antigen of expressing in silkworm detects
Get " O " type red corpuscle of people and preserve in A Shi liquid, with the physiological saline Washed Red Blood Cells of 20 times three times, finally red corpuscle is made into to 1% suspension with physiological saline.The physiological saline 50ul that adds sterilizing in every hole of U-shaped plate; The empty capsid antigen diluent 50ul that gets purifying with micropipet adds in the 1st hole, after mixing, after absorption 50ul, joins in the second hole, and serial dilution to the like this 10 holes, the 10th hole is drawn 50ul liquid and is discarded; The 11st hole and the 12nd hole are the physiology saline control, and negative silkworm blood is by the same procedure dilution.Every hole adds " O " type red cell suspension of 50ul 1% people, and after mixing, reaction is 45 minutes under 4 ℃, observations after the red corpuscle contrasted deposits fully.It is still positive when the rabbit hemorrhagic disease virus empty capsid antigen of preliminary purification is diluted to 100,000 times.
(3) Electronic Speculum and colloid gold immune electron microscopic observation
10 times of samples with water dilutions after virus-like particle collection and purifying, put copper mesh, and filter paper is used ddH after blotting liquid 2o blots with filter paper after dripping and washing again, and copper mesh is placed on acetic acid uranium drop, and filter paper is put on new acetic acid uranium drop after blotting liquid, repeat 3 times after electron microscopic observation, as Fig. 1, can be observed rabbit hemorrhagic fever virus empty capsid particle, the size with expection, conform to.Samples with water is put copper mesh after diluting 40 times, and filter paper is used ddH after blotting liquid 2o blots with filter paper after dripping and washing again, copper mesh is placed in to about 3min on the primary antibodie drop of dilution, drip to wash and blot on the anti-drop of Radioactive colloidal gold two that is placed on dilution, drip to wash and blot rear copper mesh and be placed on acetic acid uranium drop, wash dye 3 times after electron microscopic observation, as Fig. 2, can be observed absorption Radioactive colloidal gold rabbit hemorrhagic fever virus empty capsid particle, quantity is many, shows that rabbit hemorrhagic fever virus albumen VP60 can independently be assembled into the virus hollow capsid particle in Silkworm, Bombyx mori.
(4) animal immune experiment
Experimental procedure, with embodiment 1, is test group by the purifying antigen RHDV viral capsid particle of collection through intramuscular injection and 10 rabbit of each immunity of oral route, and what separately establish 5 rabbit injection adjuvants and feeding antigen is control group, and rabbit is the RHDV feminine gender.The RHDV empty capsid antigen of 5 micrograms mixes the trial-production vaccine with oily adjuvant equal-volume, 1ml/ only, in the enterprising action thing test of rabbit, gets blood after 21 days, carries out the detection of ELISA antibody titer, test group all produces the antibody for RHDV, and the injection group detects to tire and can reach more than 3200 times; Empty capsid particle by preliminary purification, amount by every rabbit 50 μ g is mixed with the propolis containing 25%-35% lipid acid and 8%-12% of same volume, optimum concn is 30% lipid acid and 10% propolis (it is 3% that lipid acid consists of palmitinic acid 8%, oleic acid 19%, other composition), after ultrasonic emulsification, pour into, after one month, oral group of detection IgA tires and also reaches 1600 times.Test group 100% produces protection antibody as a result, and control group does not detect antibody; Attacked poison experiment when getting blood, though the injection of immune group or orally all there is no rabbit death, and control group is attacked all death in latter 3 days of poison, shows typical rabbit hemorrhagic fever symptom.

Claims (5)

1. a rabbit hemorrhagic fever virus capsid protein VP60 optimized gene, is characterized in that, its nucleotide sequence is as shown in SEQ ID No.2.
2. a recombinant baculovirus, it is characterized in that, the baculovirus transfer vector DNA that comprises rabbit hemorrhagic fever virus capsid protein VP60 gene or the described optimized gene of claim 1 by use is recombinated with baculovirus DNA or swivel base obtains after reacting, wherein, described recombinant baculovirus is the recombinant bombyx mori nuclear polyhedrosis virus rBmNPV that comprises rabbit hemorrhagic fever virus capsid protein VP60 gene, or the recombinant bombyx mori nuclear polyhedrosis virus rBmNPV that comprises the described optimized gene of claim 1.
3. an insect bio-reactor for preparing rabbit hemorrhagic fever virus capsid protein VP60, it is characterized in that, the recombinate shape virus infection insect host of expressing rabbit hemorrhagic fever virus capsid protein VP60 gene or the described optimized gene of claim 1 by use obtains, wherein, described recombinant baculovirus is the recombinant bombyx mori nuclear polyhedrosis virus rBmNPV that comprises rabbit hemorrhagic fever virus capsid protein VP60 gene, or the recombinant bombyx mori nuclear polyhedrosis virus rBmNPV that comprises the described optimized gene of claim 1.
4. the preparation method of a rabbit hemorrhagic fever virus empty capsid antigen, is characterized in that, said method comprising the steps of:
1) build the baculovirus transfer vector that comprises rabbit hemorrhagic fever virus capsid protein VP60 gene or the described optimized gene of claim 1;
2) the transfer expression vector and the baculovirus that structure are obtained carry out cotransfection, obtain recombinant baculovirus, wherein, described recombinant baculovirus is the recombinant bombyx mori nuclear polyhedrosis virus rBmNPV that comprises rabbit hemorrhagic fever virus capsid protein VP60 gene, or the recombinant bombyx mori nuclear polyhedrosis virus rBmNPV that comprises the described optimized gene of claim 1;
3), by the recombinate shape virus infection insect host cell, described insect host is silkworm;
4) cultivate infected insect host, express corresponding rabbit hemorrhagic fever virus capsid antigen particle, results the expressed antigen of purifying.
5. method according to claim 4, is characterized in that,
Described insect host is silkworm larva and pupa, by silkworm larva or the pupa in recombinant Bombyx mori baculovirus infected silkworm cell or percutaneous puncture-inoculation 1-5 age, and body fluid or the tissue homogenate of collector's silkworm larva or pupa after infecting 3-6 days.
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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101215576A (en) * 2008-01-18 2008-07-09 江苏省农业科学院 Rabbit viral haemorrhagic virus capsid protein gene recombination baculovirus and bacterin

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
兔出血症病毒衣壳蛋白VP60在杆状病毒表达系统中的表达及细胞内定位;陈柳等;《浙江农业学报》;20101231;第22卷(第2期);135-139 *
酶联免疫吸附化学发光检测兔出血热病毒衣壳蛋白VP60;陈璐等;《分析化学》;20091031;第37卷;增刊 *
陈柳等.兔出血症病毒衣壳蛋白VP60在杆状病毒表达系统中的表达及细胞内定位.《浙江农业学报》.2010,第22卷(第2期),135-139.
陈璐等.酶联免疫吸附化学发光检测兔出血热病毒衣壳蛋白VP60.《分析化学》.2009,第37卷增刊.

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