CN102304529A - Preparation method of rabbit hemorrhagic fever virus empty capsid antigen - Google Patents
Preparation method of rabbit hemorrhagic fever virus empty capsid antigen Download PDFInfo
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- CN102304529A CN102304529A CN201110254439A CN201110254439A CN102304529A CN 102304529 A CN102304529 A CN 102304529A CN 201110254439 A CN201110254439 A CN 201110254439A CN 201110254439 A CN201110254439 A CN 201110254439A CN 102304529 A CN102304529 A CN 102304529A
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- hemorrhagic fever
- silkworm
- rabbit hemorrhagic
- fever virus
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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
Technical field
The present invention relates to the genetically engineered field, relate in particular to the antigenic preparation method of a kind of rabbit hemorrhagic fever virus hollow capsid.
Background technology
Rabbit hemorrhagic disease is commonly called as the rabbit pest; Be 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, enjoyed the concern of rabbit keeping because of bring enormous economic loss to rabbit keeping.Chinese reported first in 1984 should disease.1989, OIE (OIE) should disease formally classify the category-B transmissible disease as, and China classifies them as two types of 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, no cyst membrane, diameter 32-36nm; 20 body symmetries (Zhang Lihong etc., Guangdong animal and veterinary science and technology, 31:9-11; 2006), its capsid is made up of the cylindric capsomere of 32 high 5-6nm, is made up of the multiple copy of primary structure albumen VP60.Negative staining Electronic Speculum surface has the typical cup-shaped morphological structure of calicivirus, and also visible minority does not have the virus hollow capsid of core under the Electronic Speculum.
The RHDV genome contains 2 open reading frame; 5 ' terminal one 2344 amino acid whose polyprotein precursor of long open reading frame (ORF1) coding; It further is decomposed into capsid protein and a plurality of Nonstructural Protein by virus protease; Wherein capsid protein is the primary structure albumen of virus; Be called VP60, directly related with the immune response that the inducing anti-disease poison infects.3 ' terminal short open reading frame frame (ORF2), between 7025-7378bp, another little constituent of coding virus is called VP10.
The N-end of VP60 is formed the inner area of capsid protein, and the C-end is formed the outside of capsid protein.Amino acid between the 31-250 of N-end is main anti-infectious immunity determining area.Discover VP60 is proteic; During this capsid protein of vivoexpression; Under the condition that does not have other any compositions to exist; Can aggregate into naturally 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, use virus-like particle and more and more receive numerous scientists' attention as the antigen vectors of vaccine.Virus-like particle is the particle form characteristics of simulated virus; The exogenous genetic fragment product is presented to the virion surface and the chimeric particle of formation; 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 the surface at oneself, thereby in the vaccine research of pathogenic agent, play an important role.VLPs often can induce generation than inactivated vaccine and soluble polypeptide intensive humoral immunoresponse(HI) more; Because glycoprotein antigen is presented with the noninfective graininess simulation natural antigen process of presenting in its surface; And the immunne response that this method offers to cause is better than dissolved state; So the immunne response that the glycoprotein antigen that in provide protection, plays an important role causes is expected more near natural infection; Like this, virus-like particle more likely is widely used in the development of vaccine.And VLPs is not owing to wrap up nucleic acid, not reproducible; Therefore also not having 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 that RHDV can not breed on the chicken embryo, also be difficult to stable propagation in various former generations or passage cell.Present widely used vaccine is a tissue inactivation seedling.Recently, the research of new generation vaccine mainly concentrates in the development of gene engineering vaccine, in 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 to have the part same antigen and not induce the generation protective immunity with natural VP60 as VP60 during with the beta-galactosidase enzymes expressing fusion protein; In the expression system that with the t7 rna polymerase is the basis, then can express with the very similar protein of natural VP60 antigenicity and also can induce the effective protective immunity of generation, 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. efficiently express the VP60 of Spanish isolate AST/89 in yeast, expressing quantity has carried out glycosylation modified up to 1.5g/L and about 70% expressing protein; Yan Weiwei etc. insert pPICZ B with RHDV VP60 gene to have the blood clotting characteristic and can be suppressed (Yan Weiwei etc., Chinese animal doctor's journal, 23:447-449,2003) by the hyper-immune serum of anti-RHDV through the recombinant protein of Pichia anomala expression.Fernandez-Femandez etc. have adopted carrier successful expression that plum pox potyvirus makes up 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); Employing potatos such as Castanon are expressed the extract that contains VP60 rabbit are carried out immunity; 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 relatively shortcoming of immune effect, and its application prospect is also pessimistic; Recombinant viral vaccine exists to the genetically modified organism safety problem of environment diffusion; Adopt the expression level of transgenic plant production VP60 at present also undesirable; Thereby development RHD safety, new generation vaccine is imperative efficiently.
The rabbit pest are hemorrhage with respiratory system, and organa parenchymatosum's oedema, extravasated blood and the hemorrhage characteristic that is changed to, infected rabbits are everlasting dead in 48-72 hour.Can make tentative diagnosis according to lesion characteristic, make a definite diagnosis, mainly with blood clotting experiment (HA), enzyme linked immunosorbent assay (ELISA), immunoelectronmicroscopy and molecular biology method in conjunction with the virus antigen that the laboratory is detected in the rabbit tissue of dying of illness.
Baculovirus expression carrier 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 obtain expressing 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 with native protein; B: silkworm baculovirus can hold bigger foreign gene and not influence itself propagation; C: use polyhedrin gene promoter expression alien gene in late period in evening,, also do not influence expression level even recombination product pair cell is toxic; D: borrow polynary expression vector or borrow several different recombinant virus coinfections, can express 2 or more a plurality of foreign protein simultaneously, the structure and the function of supramolecule assembling of research peptide chain and albumen oligomer; E: baculovirus does not have pathogenicity bo to vertebrates, and silkworm does not have the disease with infecting both domestic animals and human, and being considered on the 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 on medical science, veterinary science and agricultural since exploitation in 1984.1997, Hubei Province, field etc. were carrier with the BmNPV that modifies, 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, expression product has good external activity.Nineteen ninety, Chu Ruiyin etc. have obtained expression to the HBsAg gene respectively 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.Tens of kinds of animal virus antigen proteins such as foot and mouth disease virus are successively expressed and purifying, and wherein quite a few is carrying out commercialized development (Li Zhiyong etc., Plos one, e2273,2008).The prevention reorganization IL preparation (Intercat) of the pet (cat and dog) that produces with the BmNPV system produce by Japan " TORAY " Co., Ltd. and put goods on the market (Kimura is grown, insect biological factory [M], Japan: census of manufacturing meeting, 2000,124-127).
The relevant technologies of baculovirus expression vector system is ripe relatively 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 rabbit hemorrhagic fever virus hollow 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 accomplished the present invention in order to address the above problem.
The objective of the invention is to overcome the deficiency of prior art, provide and utilized baculovirus expression vector system in insect body, to express the antigenic method of rabbit hemorrhagic fever virus hollow capsid safely, efficiently.
Another object of the present invention provides rabbit hemorrhagic fever virus capsid protein VP60 optimized gene.
A further object of the present invention provides 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 provides the antigenic insect bio-reactor of preparation rabbit hemorrhagic fever virus hollow capsid.
A further object of the present invention provides the rabbit hemorrhagic fever virus hollow capsid antigen through method for preparing.
A further object of the present invention provides rabbit hemorrhagic fever virus hollow capsid antigen through method for preparing as the application of rabbit hemorrhagic fever vaccine.
According to rabbit hemorrhagic fever virus capsid protein VP60 optimized gene of the present invention, its nucleotide sequence is 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 through using above-mentioned baculovirus transfer vector to infect the recombinant baculovirus that baculovirus obtains.
The present invention also provides preparation rabbit hemorrhagic fever virus hollow capsid antigenic insect bio-reactor, obtains through using above-mentioned recombinate shape virus infection insect host.
Therefore, may further comprise the steps according to the antigenic preparation method of rabbit hemorrhagic fever virus hollow capsid of the present invention:
1) making up 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 take place, obtain recombinant baculovirus;
3) with the recombinate shape virus infection insect host cell;
4) cultivate infected insect host, express corresponding rabbit hemorrhagic fever virus hollow capsid antigen, results and the expressed antigen of purifying.
According to the antigenic preparation method of rabbit hemorrhagic fever virus hollow capsid of the present invention; Wherein, The original series of described rabbit hemorrhagic fever virus capsid protein VP60 gene and the nucleotide sequence after codon optimized 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.
Antigenic preparation method comprises according to rabbit hemorrhagic fever virus hollow capsid of the present invention; 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, the carrier of pVL1393 baculovirus delivery more preferably.
According to the antigenic preparation method of rabbit hemorrhagic fever virus hollow capsid of the present invention; Wherein, constructed transfer vector is the carrier pVL1393-RHDV-VP60 that has a rabbit hemorrhagic fever virus capsid protein VP60 original series and the carrier pVL 1393-RHDV-VP60-O that has rabbit hemorrhagic fever virus capsid protein VP60 gene sequence after codon optimized.
According to the antigenic preparation method of rabbit hemorrhagic fever virus hollow capsid 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 preferred implementation 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 antigenic preparation method of rabbit hemorrhagic fever virus hollow capsid of the present invention; Wherein, Described insect host is selected from lepidopterous insects, like 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.
According to the antigenic preparation method of rabbit hemorrhagic fever virus hollow capsid of the present invention; Wherein, Described infection is meant that recombinant baculovirus is through eating or seeing through insect larvae or the pupal cell that epidermis infects 1-5 age (more preferably: with the silkworm larva or the pupa in recombinant silkworm baculovirus infection bombyx mori cell or percutaneous puncture-inoculation 1-5 age, after infecting 3-6 days, collect body fluid or the tissue homogenate that contains antigenic silkworm larva of rabbit hemorrhagic fever virus hollow capsid or pupa); Wherein, described pupal cell optimum is 1-2 days an early stage tender pupa.
The present invention adopts gene recombination technology, the capsid protein gene sequence of rabbit hemorrhagic fever virus (RHDV) is carried out codon optimized, will this proteic original series (SEQ ID NO:1) and optimize after sequence (SEQ ID NO:2) be cloned into baculovirus transfer vector (like 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) on; In the polyhedron promotor; Under p10 promotor or other virus and the control of Eukaryotic strong promoter; Through in the body or external (in vivo/in vitro) reorganization, make the original series of RHDV capsid protein and optimize after sequence be incorporated on the genome of baculovirus, obtain recombinant virus; Recombinant virus can be expressed production rabbit hemorrhagic fever virus hollow capsid antigen through adding food or adopting various means to see through insect larvae or pupal cell (optimal time is 1-2 days an early stage tender pupa) that epidermis infects 1-5 age (optimal time was four or five ages).
According to a preferred embodiment of the present invention, the rabbit hemorrhagic fever virus (RHDV) capsid protein gene codon optimized sequence, the original sequence of the capsid protein and the optimized sequence, the SEQ? ID? NO.1 and SEQ? ID? NO.2 in the nucleotide sequence was cloned into the baculovirus vector pVL1393 carrier, and then by in vivo recombination of rabbit hemorrhagic fever virus capsid protein gene of the original sequence of VP60 and the codon optimized sequence of VP60-O shift to the parental strain of baculovirus Bm-NPV-ZJ8 genome, Alternative Polyhedrin gene on the genome, by plaque screening and PCR detection techniques, to obtain carrying rabbit hemorrhagic fever virus capsid protein gene recombinant silkworm rod Virus rBmNPV (HDV-VP60), rBmNPV (HDV-VP60-O); their infected silkworm cell lines or puncture inoculation 1-5 instar silkworm larvae or pupae, blooms rBmNPV (HDV-VP60), rBmNPV (RHDV-VP60 -O); when rBmNPV (RHDV-VP60), rBmNPV (RHDV-VP60-O) in the silkworm body copy, VP60 and VP60-O gene polyhedrin gene (polh) promoter expressed under the control and self-assembled into Rabbit haemorrhagic fever virus empty capsid antigen; infection 3-6 days (best of five days, 25 ℃ rearing temperature) were collected with rabbit hemorrhagic fever virus empty capsid antigen silkworm larvae or pupae humoral (or whole homogenate ), after exposure to radiation to kill baculovirus protein purification after the safe and efficient air rabbit hemorrhagic fever virus capsid antigen, the antigen can be used to prepare the prevention of rabbit hemorrhagic disease virus injectable vaccine.
The inventive method adopts baculovirus expression system in silkworm biological reactor, to produce rabbit hemorrhagic fever virus antigen safely, efficiently; Its production cost significantly is lower than traditional antigenic method of preparation rabbit hemorrhagic fever virus; Found the factory, the three wastes, electric power and the consumption of water resources equal energy source are few.Because silkworm has been a food medicine dual-purpose insect by the approval of China Ministry of Health, so behind the antigen purification that the inventive method is prepared, security is high, can directly make the vaccine immunity animal.
In general, the inventive method can reduce the antigenic production cost of rabbit hemorrhagic fever virus hollow capsid significantly, has plurality of advantages such as safety, efficient, less energy consumption, cost are low.
Description of drawings
Fig. 1 is the electron-microscope scanning figure of the RHDV hollow capsid particle of silkworm production;
The hollow capsid immuno-gold labeling electron-microscope scanning figure that Fig. 2 produces for silkworm.
Embodiment
Produce the present invention in detail below in conjunction with embodiment and accompanying drawing, those having ordinary skill in the art will appreciate that these instances are illustrative fully, do not limit protection scope of the present invention.
Experiment material
E. coli strains E.coli DH
5 αAvailable from Promega company; Cloning vector pEASY-T3 is available from full formula King Company; Cloning vector pMD18-T is available from TaKaRa company; Prokaryotic expression carrier pET-28a
+, recipient bacterium E.coli TOP10 and BL21 (DE3) be conventional bacterial strain available from Novagen company, delivery carrier pVL1393 is available 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, synthetic the obtaining of sequence VP60-O direct gene after codon optimized; 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: used restriction enzyme and supporting damping fluid are all available from Promega company; T4 DNA Ligase and damping fluid are the Promega Company products; LA Taq polysaccharase and damping fluid are available from TaKaRa company; RnaseA, dNTPs are available from Sigma company; The DNA of all size and protein molecular weight standard are TranGen Biotech Company products; DEPC, M-MLV-Rtase (reversed transcriptive enzyme) are available from Promega company.
Biochemical reagents: bisacrylamide, acrylamide, IPTG, X-Gal are available 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 available from Sigma company; Agarose is the Sunbiotech Company products; Yeast extract (Yeast Extract), Tryptones are all available from Britain OXOID company; 0.2um, the 0.45um filter is available from Gelman Sciences company; Ethidium bromide (EB), Coomassie brilliant blue R-250 are available from Fluka company; Agar powder is Japanese import packing; Ni-NTA resin, Proteinase K, foetal calf serum are available from Invitrogen company; Other is homemade or the import analytical reagent.Primer is synthetic by three rich polygala root biotechnology limited liability companys
Substratum: the intestinal bacteria substratum 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 through the method for RT-PCR.The reverse transcription special primer that designs is: RHDV-RT:5 '-GGATTAAAACCTAACCTACC-3 '.The amplimer of the original series VP60 of capsid protein gene is: the VP60 upper reaches: 5 '-G
GAATTCAACATGGAGGGCAAAGCCCGCACAG-3 ' (EcoR I); The VP60 downstream: 5 '-GA
AGATCTTCAGACATAAGAAAAGCCATTG-3 ' (Bgl II).
After pcr amplification finishes, analyze clip size through agarose gel electrophoresis.Reclaim target gene fragment, be connected (pMD18-T) with cloning vector and connect.Transformed E .coli heat shock competent cell is identified 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; After 220r/min shakes and trains the back of spending the night; Draw the fresh bacterium liquid of 1mL to aseptic Eppendorf pipe; Add a small amount of glycerine; To seal after film seals; Three order-checking portions of rich Bioisystech Co., Ltd check order to Beijing, and the result is shown in SEQ ID NO.1, and aminoacid sequence is shown in SEQ ID NO.3.Sequencing result is analyzed sequence with DNAStar, DNAMAN software.
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 the enzyme switchback is received with cut through EcoR I/Bgl II double digestion enzyme after being connected of transfer vector pVL1393.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 products explanation preparing culture medium, cultivate bombyx mori cell BmN down for 27 ℃.With the about 50ml of cell of Bombyx mori nuclear polyhydrosis virus parent plant Bm-NPV-ZJ8 infection logarithmic phase, infection multiplicity is to collect virus infection liquid after 1,3～4 days; Centrifugal (10000rpm * 10min); Remove deposition, supernatant centrifugal 1 hour with 25000rpm removes supernatant; (contain Tris 12.1g among the 1000ml with 1ml viral DNA extract; EDTA 33.6g, KCl 14.1g, pH7.5) suspension virus particle deposition; Extracting DNA, it is subsequent use to put 4 ℃ of preservations.
4, structure and the acquisition of recombinant silkworm baculovirus rBmNPV (RHDV-VP60)
Inoculate about 1 * 10
6Cell behind the cell attachment, is removed and is contained foetal calf serum (FBS) substratum in the 15cm2 culturing bottle, gives a baby a bath on the third day after its birth time with the substratum that does not contain FBS, and adding 1.5ml does not have the FBS substratum.In a sterile tube, add 1 μ g silkworm baculovirus parent plant Bm-NPV-ZJ8DNA successively; 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; Mixing gently; After leaving standstill 15min, dropwise join and carry out cotransfection in the culturing bottle.Microscopic examination will not contain polyhedrosis plaque and pick out, and repeat above step, obtain pure recombinant silkworm baculovirus rBmNPV (RHDV-VP60) through 2～3 purifying of taking turns.
With the BmN cell of recombinant silkworm baculovirus rBmNPV (RHDV-VP60) infection normal growth, cultivate and collect supernatant liquor after 3 days, promptly contain a large amount of recombinant virus rBmNPV (RHDV-VP60) in the supernatant.
Utilize PCR method to analyze exogenous origin gene integrator, Oligonucleolide primers is:
The VP60 upper reaches: 5 ' G
GAATTCAACATGGAGGGCAAAGCCCGCACAG-3 ' (EcoR I); The VP60 downstream: 5 '-GA
AGATCTTCAGACATAAGAAAAGCCATTG-3 ' (Bgl II).
The result proves and has obtained recombinant virus.
6, RHDV-VP60 efficiently expresses in silkworm pupa and silkworm body
Used silkworm pupa is JY1 for the high expression level kind.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.Behind the first feeding 48h select the identical silkworm of mean body weight and cocoond seven days after 15 identical silkworm chrysalises of mean body weight, every silkworm chrysalis and silkworm inoculate about 1.0 * 10
5RBmNPV (RHDV-VP60) collects the morbidity silkworm chrysalis and gets silkworm blood after 4-5 days ,-20 ℃ frozen to carry out the detection of double-antibody sandwich elisa method.
7, the collection of RHDV-VP60 virus-like particle and purifying.
To contain express have the silkworm chrysalis of RHDV-VP60 in homogenizer, to grind with the PBS (solid-liquid ratio is 1: 9) of precooling after, filter with the 0.45um filter.In 30% sucrose solution, 1.5 * 10
5Centrifugal 2 hours of g ultra-high speed.Solution with the Tris-HCl (PH7.0) that contains 0.1M NaCl redissolves deposition to original volume, crosses cation-exchange chromatography filler SP (GE company), the Tris-HCl of 0.5M NaCl (PH7.0) wash-out.Again through sieve chromatography S200 (GE company).Purity can reach 95%, and yield can reach more than 40%.Prove that simultaneously the target protein of in silkworm, expressing can be self-assembled into the virus hollow capsid particle under high density, but also has set up the antigenic purification process of corresponding silkworm expression genetically engineered rabbit hemorrhagic disease virus hollow capsid.
8, the detection of expression product
(1) ELISA and Western blotting detect
Silkworm behind the injecting virus is collected silkworm blood when waiting to fall ill.The ELISA method detects the height of RHDV-VP60 antigen protein expression amount; Coating buffer is as blank; As negative control, the rabbit anteserum after the RHDV-FR2 protein immunization new zealand white rabbit of prokaryotic expression is a first antibody with normal silkworm blood, and the goat anti-rabbit antibody of HRP mark is a second antibody.Appoint and to get the sample in the silkworm blood of receiving, do gradient dilution with coating buffer, from 50 * twice doubling dilution to 6400 times, the diplopore detection is done at each gradient place, averages during calculating, respectively gets 100 μ L and encapsulates on the enzyme plate.Result such as table 1 show that RHDV-VP60 gene expression amount in silkworm is high, and the expression amount in average every boss silkworm or the silkworm chrysalis is 1mg at least, under 3200 times of dilutions even greater dilution, still can detect the specific reaction of antigen and antibody.
Table 1ELISA detects the VP-60 gene that silkworm biological reactor is expressed
Western blotting result is illustrated in the specific band that can detect the 60kD size in the supernatant liquor of silkworm hemolymph sample behind the recombinant virus infection.
The antigenic blood clotting experiment of the rabbit hemorrhagic disease virus hollow capsid of (2) expressing in the silkworm detects
Get " O " type red corpuscle of people and in A Shi liquid, preserve,, at last red corpuscle is made into 1% suspension with physiological saline with 20 times physiological saline Washed Red Blood Cells three times.The physiological saline 50ul that adds sterilization in every hole of U-shaped plate; The virus hollow capsid solution dilution liquid 50ul that contains that gets the suitable multiple dilution with micropipet adds in the 1st hole, behind the mixing, joins in second hole behind the absorption 50ul, and serial dilution to the 10 holes like this, 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 50ul1% people, and behind the mixing, 4 ℃ were reacted 45 minutes down, and red corpuscle to be contrasted deposits the back observations fully.During the rabbit hemorrhagic disease virus hollow capsid antigen diluent to 64000 of preliminary purification times for still positive.
(3) animal immune experiment.
Is test group with the purifying antigen RHDV viral capsid particle of collecting through 10 rabbit of each immunity of intramuscular injection and oral route, and other establishes 5 rabbit injection adjuvants and feeding antigenic is control group, and rabbit is the RHDV feminine gender.5ug RHDV hollow 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 ELISA antibody titer and detects; Test group all produces the antibody to RHDV, and injection groups detects to tire and can reach more than 3200 times; Empty capsid particle with preliminary purification; Amount by every rabbit 50 μ g is mixed with the propolis that contains 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); Behind 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; When getting blood, attack poison experiment, all do not have rabbit dead, and control group is attacked all death in back 3 days of the poison, shows typical rabbit hemorrhagic fever symptom no matter the injection of immune group is still oral.
Embodiment 2, the rabbit hemorrhagic fever virus capsid protein gene solubility expression of sequence VP60-O in silkworm biological reactor and the detection of expression product after codon optimized
1, optimizes the acquisition of back sequence VP60-O
According to the silkworm codon preference rabbit hemorrhagic fever viruse capsid protein gene original series VP60 that embodiment 1 records is optimized; Do not change amino acid sequence; Optimize back sequence VP60-O shown in SEQIDNO.2; The rare codon that contains series connection in the original series; This reduced translation sequences or even remove translating equipment; Optimize back sequence CAI value and rise to 0.79 by 0.72; GC content and unsuitable peak have been adjusted to prolong the half-life of mRNA; GC content is adjusted into 53.34% by 54.9%; Those influence mRNA loop-stem structure stable and that combine with ribosomes and are destroyed; In 90 sites the BamHI restriction enzyme site is arranged in the original series; There is the XhoI restriction enzyme site in 487 sites, and the sequence after the optimization does not have this two restriction enzyme sites.Sequence VP60-O behind directly synthetic the optimization also is cloned on the carrier pUC57, and two ends have Bam HI, EcoR I restriction enzyme site respectively.Order-checking identify after analyze SEQ ID NO:2 and amino acid sequence corresponding 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.
With being connected of the transfer vector pVL1393 of plasmid pUC57-RHDV-VP60-O behind the product of Bam HI/EcoR I double digestion and Bam HI/EcoR I double digestion.
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 silkworm baculovirus rBmNPV (RHDV-VP60-O)
With embodiment 1.
With the BmN cell of recombinant silkworm baculovirus rBmNPV (RHDV-VP60-O) infection normal growth, cultivate and collect supernatant liquor after 3 days, promptly contain a large amount of recombinant virus rBmNPV (RHDV-VP60-O) in the supernatant liquor.Utilize PCR method to analyze exogenous origin gene integrator.Oligonucleolide primers is: identify sequence by two primers of the VP60-O gene order indoor design of optimizing:
RHDV-F：5′-ACCTTGGTCCTTTCCGTATATAA-3′；
RHDV-R：5′-CTTGGCGACAGTTTGAGCAC-3′。
The result proves and has obtained recombinant virus.
5, RHDV-VP60-O efficiently expresses in silkworm pupa and silkworm body
Used silkworm pupa is JY1 for the high expression level kind.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.Behind the first feeding 48h select the identical silkworm of mean body weight and cocoond seven days after 15 identical silkworm chrysalises of mean body weight, every silkworm chrysalis and silkworm inoculate about 1.0 * 10
5RBmNPV (RHDV-VP60-O) collects the morbidity silkworm chrysalis and gets silkworm blood after 4-5 days ,-20 ℃ frozen to carry out the detection of double-antibody sandwich elisa method.
6, the collection of RHDV-VP60-O virus-like particle and purifying
To contain express have the silkworm chrysalis of RHDV-VP60-O in homogenizer, to grind with the PBS (solid-liquid ratio is 1: 9) of precooling after, filter with the 0.45um filter.In 30% sucrose solution, 1.5 * 10
5Centrifugal 2 hours of g ultra-high speed.Solution with the Tris-HCl (PH7.0) that contains 0.1MNaCl redissolves deposition to original volume, crosses cation-exchange chromatography filler SP (GE company), the Tris-HCl of 0.5M NaCl (PH7.0) wash-out.Again through sieve chromatography S200 (GE company).Purity can reach 95%, and yield can reach more than 40%.Prove that simultaneously the target protein of in silkworm, expressing is soluble, but also has set up the antigenic purification process of corresponding silkworm expression genetically engineered rabbit hemorrhagic disease virus hollow capsid.
7, rabbit hemorrhagic disease virus detects the preparation of antibody
The capsid protein sequence VP60-O that will design after three pairs of special primers will be optimized divides three sections to carry out prokaryotic expression, and design of primers is following:
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
With plasmid pUC57-RHDV-VP60-O is template, uses above-mentioned three couples of primer amplification purpose fragment RHDV-FR1, RHDV-FR2, RHDV-FR3 respectively; Glass milk method purifying and recovering PCR product; The PCR purified product is connected with cloning vector pEASY-T3; Connect product and transform the intestinal bacteria thermal shock competent cell that has prepared; Choose spot cultivation the carrying out evaluation of recombinant plasmid; The bacterial strain that picking plasmid band is stepped back after the cytoclasis method Rapid identification; Alkaline lysis method of extracting recombinant plasmid pT3-RHDV-FR1, pT3-RHDV-FR2 and pT3-RHDV-FR3; Carry out double digestion with Bam HI/Hind III and identify, and enzyme is cut the correct reorganization bacterium bacterium liquid of evaluation send three rich polygala root companies to carry out the dna sequencing checking.Sequencing result and the sequence of previous plasmid pUC57-RHDV-VP60-O are carried out the nucleotide sequence comparison, and the result shows three aim sequences in the recombinant plasmid and before the plasmid sequence result was consistent, phase shift mutation does not take place and contain the restriction enzyme site of 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 and recovering enzyme is cut product; Be connected to by on the expression vector pET-28a of Bam HI/Hind III double digestion digestion; Connect product and transform the Top10 competent cell; Carry out cytoclasis method Rapid identification earlier after choosing spot; Then a small amount of extracting recombinant plasmid pET28a-RHDV-FR1, pET28a-RHDV-FR2, pET28a-RHDV-FR3 fast of alkaline process identifies 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 the BL21 competent cell respectively; IPTG collects bacterium liquid after inducing 4h; With SDS-PAGE electrophoretic analysis expression, result's demonstration is compared with negative control, and the expressing fusion protein band that transforms the bacterial strain appearance of recombinant plasmid pT3-RHDV-FR2 concentrates most.Induce after the best expression strain list bacterium colony shaking culture of picking effect, after ultrasonication, the SDS-PAGE electrophoretic analysis shows that the fusion rotein (His-RHDV-FR2) of expressing is present in the deposition, 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; Related solution and method that adopt to handle inclusion body protein with solubilization of inclusion bodies after; 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, and SDS-PAGE analyzes and confirms combination of proteins situation, the distribution situation of target protein in elutriant.The result shows the single band that size is correct; The target protein His-RHDV-FR2 size of expressing is consistent with positive control, and when carrying out wash-out with urea NTA-100, elution peak is maximum; Cut off purifying band albumen with the sterilization pocket knife behind the 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; Method is the database search identification method of 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 greater than 50; AA sequence that can detect and expected sequence matching rate are exactly the expression product of target gene RHDV-FR2 up to 34.68% so can confirm purifying protein.The fusion rotein that purifying is expressed also carries out albumen and concentrates, and adopts the Bradford method to survey the concentration that concentrates the back 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, send after micelle is ground in Chinese Academy of Sciences's heredity with grow institute, the negative rabbit of immune RHDV prepares polyclonal antibody.
(5) the polyclonal antibody detection of tiring
Obtain polyclonal antibody behind the RHDV-FR2 protein immunization new zealand white rabbit of purifying 4 times; Fusion rotein with purifying is made envelope antigen; Doing one with the polyclonal antiserum of PBS dilution different multiples 100,200,400,800,1600,3200,6400,12800,25600,51200 resists; The sandwich ELISA detection method is surveyed antibody titer figure; Rabbit anteserum is made negative control before the immunity; The light absorption value at the A492nm place is an X-axis with each extension rate, and the light absorption value mean value under each extension rate is Y-axis.Usually greater than 2.1 times of the negative control OD value of regulation, promptly positive (calculating) with zeroing back, blank hole, result's demonstration: tiring of antibody can reach 1: 3200 when homemade antibody was 10 μ g/ml in antigen protein concentration.
8, the detection of expression product
(1) ELISA and Western blotting detect
Silkworm behind the injecting virus is collected silkworm blood when waiting to fall ill.The ELISA method detects the height of RHDV-VP60-O antigen protein expression amount; Coating buffer is as blank; As negative control, the rabbit anteserum after the RHDV-FR2 protein immunization new zealand white rabbit of prokaryotic expression is a first antibody with normal silkworm blood, and the goat anti-rabbit antibody of HRP mark is a second antibody.Appoint and to get the sample in the silkworm blood of receiving, do gradient dilution, from 100 * twice doubling dilution to 6400 times with coating buffer.Do diplopore and detect at each gradient place, respectively gets 100 μ L and encapsulate on the enzyme plate.Result such 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, under 6400 times of dilutions even greater dilution, still can detect the specific reaction (as shown in table 2) of antigen and antibody.
Table 2ELISA detects the VP60-O gene of expressing in the silkworm biological reactor
Western blotting result is illustrated in the specific band that can detect the 60kD size in the supernatant liquor of the hemolymph sample of silkworm behind the recombinant virus infection.
The antigenic blood clotting experiment of the rabbit hemorrhagic disease virus hollow capsid of (2) expressing in the silkworm detects
Get " O " type red corpuscle of people and in A Shi liquid, preserve,, at last red corpuscle is made into 1% suspension with physiological saline with 20 times physiological saline Washed Red Blood Cells three times.The physiological saline 50ul that adds sterilization in every hole of U-shaped plate; The hollow capsid antigenic dilution 50ul that gets purifying with micropipet adds in the 1st hole, behind the mixing, joins in second hole behind the absorption 50ul, and serial dilution to the 10 holes like this, 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 behind the mixing, 4 ℃ were reacted 45 minutes down, and red corpuscle to be contrasted deposits the back observations fully.Still positive during ten thousand times of the rabbit hemorrhagic disease virus hollow capsid antigen diluents to 10 of preliminary purification.
(3) Electronic Speculum and colloid gold immune electron microscopic observation
Copper mesh is put in 10 times of samples with water dilutions behind virus-like particle collection and the purifying, 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 the acetic acid uranium drop, and filter paper is put on the new acetic acid uranium drop after blotting liquid, repeat 3 times after electron microscopic observation, like Fig. 1, can be observed rabbit hemorrhagic fever virus hollow capsid particle, the size conform to expection.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 about 3min on the anti-drop of dilution; Drip to wash and blot on the Radioactive colloidal gold two anti-drops that are placed on dilution; Drip to wash and blot the back copper mesh and place on the acetic acid uranium drop; Wash dye 3 times after electron microscopic observation, like Fig. 2, can be observed absorption Radioactive colloidal gold rabbit hemorrhagic fever virus hollow capsid particle; Quantity is many, shows that rabbit hemorrhagic fever virus albumen VP60 can independently be assembled into the virus hollow capsid particle in the silkworm body.
(4) animal immune experiment
Experimental procedure is with embodiment 1, is test group with the purifying antigen RHDV viral capsid particle of collecting through 10 rabbit of each immunity of intramuscular injection and oral route, and other establishes 5 rabbit injection adjuvants and feeding antigenic is control group, and rabbit is the RHDV feminine gender.The RHDV hollow 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 ELISA antibody titer and detects; Test group all produces the antibody to RHDV, and injection groups detects to tire and can reach more than 3200 times; Empty capsid particle with preliminary purification; Amount by every rabbit 50 μ g is mixed with the propolis that contains 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); Behind 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; When getting blood, attack poison experiment, all do not have rabbit dead, and control group is attacked all death in back 3 days of the poison, shows typical rabbit hemorrhagic fever symptom no matter the injection of immune group is still oral.
Claims (10)
1. a rabbit hemorrhagic fever virus capsid protein VP60 optimized gene is characterized in that its nucleotide sequence is shown in SEQ ID No.2.
2. a recombinant baculovirus is characterized in that, comprises the baculovirus transfer vector DNA of rabbit hemorrhagic fever virus capsid protein VP60 gene or the said optimized gene of claim 1 and baculovirus DNA is recombinated or swivel base reaction back obtains through use.
3. recombinant baculovirus according to claim 2; It is characterized in that; Said recombinant baculovirus is the recombinant bombyx mori nuclear polyhedrosis virus rBmNPV that comprises rabbit hemorrhagic fever virus capsid protein VP60 gene, or comprises the recombinant bombyx mori nuclear polyhedrosis virus rBmNPV of the said optimized gene of claim 1.
4. an insect bio-reactor for preparing rabbit hemorrhagic fever virus capsid protein VP60 is characterized in that, obtains through using the recombinate shape virus infection insect host of expressing rabbit hemorrhagic fever virus capsid protein VP60 gene or the said optimized gene of claim 1.
5. the antigenic preparation method of rabbit hemorrhagic fever virus hollow capsid is characterized in that, said method comprising the steps of:
1) makes up the baculovirus transfer vector that comprises rabbit hemorrhagic fever virus capsid protein VP60 gene or the said optimized gene of claim 1;
2) the transfer expression vector and the baculovirus that structure are obtained carry out cotransfection, obtain recombinant baculovirus;
3) with the recombinate shape virus infection insect host cell;
4) cultivate infected insect host, express corresponding rabbit hemorrhagic fever virus capsid antigen particle, results and the expressed antigen of purifying.
6. method according to claim 5; It is characterized in that; Said baculovirus is selected from BmNPV, AcMNPV, ApNPV, HaNPV, HzNPV, LdMNPV, MbMNPV, OpMNPV, SlMNPV, SeMNPV or SpltNPV, is preferably Bombyx mori nuclear polyhydrosis virus BmNPV.
7. method according to claim 5; It is characterized in that; Described host is selected from lepidopterous insects; Be preferably 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) or gypsymoth (Lymantria dispar), more preferably silkworm.
8. method according to claim 5 is characterized in that,
Described insect host is silkworm larva and pupa, with the silkworm larva or the pupa in recombinant silkworm baculovirus infection bombyx mori cell or percutaneous puncture-inoculation 1-5 age, and the body fluid or the tissue homogenate of collector's silkworm larva or pupa after infecting 3-6 days.
9. by the rabbit hemorrhagic fever virus capsid protein VP60 of each said method preparation in the claim 5～8.
10. the said rabbit hemorrhagic fever virus of claim 9 capsid protein VP60 is as the application of rabbit hemorrhagic fever vaccine.
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| CN111575315A (en) * | 2020-05-29 | 2020-08-25 | 青岛易邦生物工程有限公司 | Rabbit viral hemorrhagic disease virus type II VLP vaccine |
| CN112480269A (en) * | 2020-12-11 | 2021-03-12 | 杭州爱谨生物科技有限公司 | Rabbit viral hemorrhagic disease virus VP10-VP60 recombinant protein and preparation method and application thereof |
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