WO2008116368A1 - Méthode de préparation d'antigènes du virus de la fièvre aphteuse - Google Patents

Méthode de préparation d'antigènes du virus de la fièvre aphteuse Download PDF

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WO2008116368A1
WO2008116368A1 PCT/CN2008/000126 CN2008000126W WO2008116368A1 WO 2008116368 A1 WO2008116368 A1 WO 2008116368A1 CN 2008000126 W CN2008000126 W CN 2008000126W WO 2008116368 A1 WO2008116368 A1 WO 2008116368A1
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baculovirus
foot
mouth disease
silkworm
recombinant
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PCT/CN2008/000126
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English (en)
Chinese (zh)
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Jixing Liu
Zhifang Zhang
Zhiyong Li
Yongzhu Yi
Xiangping Yin
Yinmei Bai
Yinv Li
Baoyu Li
Xuerui Li
Bin Yang
Xi Lan
Guifang Shen
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Lanzhou Veterinary Research Institute Chinese Academy Of Agricultural Sciences
Biotechnology Research Institute Chinese Academy Of Agricultural Sciences
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Priority to GB0916723.0A priority Critical patent/GB2463783B/en
Publication of WO2008116368A1 publication Critical patent/WO2008116368A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/125—Picornaviridae, e.g. calicivirus
    • A61K39/135—Foot- and mouth-disease virus
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C07K14/08—RNA viruses
    • C07K14/085—Picornaviridae, e.g. coxsackie virus, echovirus, enterovirus
    • C07K14/09—Foot-and-mouth disease virus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C12N15/866—Baculoviral vectors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/14011—Baculoviridae
    • C12N2710/14111—Nucleopolyhedrovirus, e.g. autographa californica nucleopolyhedrovirus
    • C12N2710/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2710/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/32011—Picornaviridae
    • C12N2770/32111—Aphthovirus, e.g. footandmouth disease virus
    • C12N2770/32122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • the invention relates to a method for preparing an antigen, in particular to a method for expressing a foot-and-mouth disease antigen in an insect by using a recombinant baculovirus, and belongs to the field of genetic engineering. Background technique
  • Foot-and-mouth disease is an acute, highly-contact, febrile infectious disease caused by Foot-and-mouth disease virus (FMDV), which is known for its rapid spread and high infection rate. Once the disease erupts, it will cause huge economic losses to the country of incidence. countries around the world attach great importance to the study of the disease, and the International Veterinary Bureau ranks it as the first class A infectious disease.
  • Foot-and-mouth disease is a member of the foot-and-mouth disease virus family of the small RNA virus family. There are 7 serotypes of A, 0, C, Asia I, SA T1, SA T2 and SA T3. At present, the prevention and control of foot-and-mouth disease in China is still based on prevention.
  • the Roxvirus Expression System was established in the 1980s. Since the first significant use of the baculovirus expression system in 1983 to express human alpha-interferon (Smith, Mol. Cell Biol" 3: 2156-2165, 1983), dozens of foreign genes have been efficiently expressed, In China alone, there are ⁇ -interferons (Yang Guanzhen et al., Journal of Biochemistry and Biophysics, 22: 355-361, 1990), and mushroom protease inhibitors (Ji et al., Sericulture Science, 21: 223-227, 1995). Marek's virus glycoprotein ⁇ (Xiao Qingli et al., Sericulture Science, 23: 104-108, 1997), etc.
  • the advantages of using this system to produce foot-and-mouth disease antigens are: 1.
  • the expression system is highly efficient, yielding Up to the level of 10 mg/worm. It can greatly reduce the production cost of foot-and-mouth disease antigen and make it possible to mass-produce the foot-and-mouth disease antigen by genetic engineering.
  • This expression system is a eukaryotic expression system, and its expression is exogenous. The protein can be post-translationally modified to provide a biochemical shield and biological activity similar to natural products, which provides a normal protein structure and biological immunological activity for the expressed foot-and-mouth disease antigen.
  • the baculovirus expression system especially the silkworm (Bm)-Bomworm baculovirus (BmNPV) expression system, is the world's most commercially valuable eukaryotic organism.
  • BmNPV silkworm-Bomworm baculovirus
  • the technical problem to be solved by the present invention is to overcome the deficiencies of the prior art and to provide a method for safely and efficiently expressing foot-and-mouth disease antigen in insects using a baculovirus expression system.
  • a method for preparing a foot-and-mouth disease antigen comprises: cloning a different gene combination of foot-and-mouth disease into a baculovirus carrier to obtain a transfer vector; transfecting the baculovirus with the obtained transfer vector for DNA recombination to obtain a recombinant baculovirus Infecting an insect host with a recombinant baculovirus; culturing the infected insect host for expression of foot-and-mouth disease antigen; harvesting and purifying the expressed antigen.
  • the different gene combinations of the foot-and-mouth disease are preferably the base sequences shown in SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 5;
  • the baculovirus carrier is preferably selected from the group consisting of AcRP23-/acZ, AcRP6-SC, AcUWl-/acZ, BacPAK6, Bac to Pac, Bacmid, BlucBacII (pETL), p2Bac, p2Blue, p89B310, pAc360, pAc373, pAcAB3, pAcAB 4, PAcAS3, pAcC129, pAcC4, DZI, pAcGP67, pAcIEl, pAcJPl, pAcMLF2, pAcMLF 7, pAcMLF 8, pAcMPK pAcMP2, pAcRP23, pAcRP 25, pAcRW4, pAcsMAG, pAcUWK pAcUW21, pAcUW2A, pAcUW2B, pAcUW3, pAc
  • the transfer vector constructed is preferably pVL1393 (P1-2A3C)>pVL1393 (ORF) or pVL1393 (VP1).
  • the baculovirus is selected from the group consisting of BmNPV, AcMNPV, ApNPV, BsSNPV, CfM PV, EoSNPV, HaNPV HzNPV, LdMNPV, MbMNPV, OpMNPV S1MNPV, SeMNPV, or TeNPV, preferably Bombyx mori Bm-NPV-ZJ8;
  • the recombinant baculovirus is preferably any one of the following: (1) Recombinant Bombyx mori nuclear polyhedrosis virus rBm PV (ORF), the microbial deposit number is: CGMCC NO. 1980; The preservation time is: March 20, 2007
  • the deposit unit is: General Microbiology Center of China Microbial Culture Collection Management Committee; The address is: No.13, Zhongguancun, Haidian District, Beijing, China, Institute of Microbiology, Chinese Academy of Sciences; (2) Recombinant Bombyx mori nuclear polyhedrosis virus rBmNPV (P1-2A3C), the microbial deposit number is: CGMCC NO.1979; : General Microbiology Center of China Microbial Culture Collection Management Committee; The preservation address is: No.13, Zhongguancun, Haidian District, Beijing, China; Institute of Microbiology, Chinese Academy of Sciences; (3) Recombinant Bombyx mori nuclear polyhedrosis virus rBmNPV (VP1), its microbial preservation No
  • the insect host is selected from the group consisting of Bombyx mori, Bombyx mandarina, Philosamia cynthia ricim, Dictyoploca japanica, Philosamia cynthia pryeri, Antheraea pernyi, Japanese tussah (Antheraea yamamai), Antheraea polyphymus, Atographa califorica, Ectropis obliqua, Mamestra brassicae, Spodoptera littoralis, Autumn armyworm ( Spodoptera fmgiperda ), Trichoplusia ni, Thaumetopoea wilkinsoni, Heliothis armigera, Heliothis zea, Heliothis assulta, Heliothis virescens ), Pseudaletia separata, Lymantria dispar, etc.; more preferably Bombyx mori.
  • the infection refers to a recombinant baculovirus infecting 1-5-year-old insect larvae or corpus callosum by mouth or through the epidermis (more preferably: infecting silkworm cells with recombinant silkworm baculovirus or inoculation 1-5 years old)
  • the silkworm larva or pupa collects body fluid or tissue pulp of the silkworm larva or cockroach containing the foot-and-mouth disease antigen after 3-6 days of infection; wherein the corpus callosum is 1-2 days of early tenderness.
  • the invention adopts genetic recombination technology to construct different gene combinations derived from foot-and-mouth disease virus, including P1-2A3C, ORF and VP1, into various baculovirus carrier vectors (such as AcRP23-lacZ, AcRP6-SC, AcUWl-lacZ, BacPAK6, Bac to Pac, Bacmid, BlueBacII (pETL), p2Bac, p2Blue, p89B310, pAc360, 373, pAcAB3, 4, pAcAS3, pAcC129, C4, DZ1, pAcGP67, pAcIEl, pAcJPl, pAcMLF2 7, 8, pAcMPl, 2, pAcRP23, 25, pAcRW4, pAcsMAG, pAcUWl, 21, 2A, 2B, 3, 31, 41, 42, 43, 51, pAcVC2, 3, pAcYMl, pAcJ
  • Recombinant viruses can infect insect larvae or carcasses of 1-5 years (optimally four or five years old) through the epidermis by oral or by various means (optimal time is 1-2 days of early tenderness) ), expressing the production of foot-and-mouth disease virus antigen.
  • a most preferred technical solution in the present invention is: inserting the base sequence represented by SEQ ID NO: 1 (P1-2A3C), SEQ ID NO: 3 (ORF) or SEQ ID NO: 5 (VPl) into a carrier
  • P1-2A3C SEQ ID NO: 1
  • ORF SEQ ID NO: 3
  • VPl SEQ ID NO: 5
  • Silkworm hemolymph can produce 10 milli FMD antigens above, after killing infection pathogens, through protein purification after safe and efficient FMD antigens, such antigens can be used to prepare vaccines.
  • the method of the invention adopts the baculovirus expression system to safely and efficiently produce the foot-and-mouth disease antigen in the silkworm bioreactor, and the production cost thereof is significantly lower than the traditional method for preparing the foot-and-mouth disease antigen (for example, preparing the foot-and-mouth disease antigen by the cell propagation virus), without investing in the establishment of the plant. There are no three wastes, and energy consumption such as electricity and water resources is extremely low. Since the silkworm has been approved by the Ministry of Health of China as a food-and-disease insect, the antigen prepared by the method of the present invention has high safety and can directly produce a vaccine-immunized animal.
  • the method of the invention can greatly reduce the production cost of foot-and-mouth disease antigen, and has the advantages of safety, high efficiency, low energy consumption, low cost and the like.
  • E. coli TGI and DH 5a were purchased from Promega; carrier vector pVL1393 (purchased from Invitrogen), silkworm cell BmN, Bombyx mori nuclear polyhedrosis virus Bm-PV-ZJ8 by Institute of Biotechnology, Chinese Academy of Agricultural Sciences
  • carrier vector pVL1393 purchased from Invitrogen
  • the foot-and-mouth disease virus was preserved by the National Reference Laboratory for Foot-and-Mouth Disease of Lanzhou Veterinary Research Institute of the Chinese Academy of Agricultural Sciences
  • the antigen detection kit was prepared by the Lanzhou Veterinary Research Institute of the Chinese Academy of Agricultural Sciences
  • the silkworm variety JY1 was preserved by the Institute of Biotechnology, Chinese Academy of Agricultural Sciences.
  • Enzymes and reagents Restriction enzymes and ligases are products of Promega.
  • Biochemical reagents IPTG and X-Gal are products of Promega.
  • Lipofectin low melting point agarose LMP, PCR kits, T 4 DNA ligase, RNA enzyme, Proteinase K, fetal calf serum, and other reagents were purchased from Invitrogen Corporation, cell culture medium TC-100 were purchased from Sigma.
  • E. coli medium was LB (1% peptone, 0.5% yeast extract, 1% NaCl, pH 7.0); the silkworm cell culture medium was TC-100.
  • the primers were designed to amplify the foot-and-mouth disease virus antigen protein P1-2A gene and the non-structural protein 3C gene by RT-PCR.
  • the designed primers for the antigenic protein P1-2A gene and the non-structural protein 3C gene are the designed primers for the antigenic protein P1-2A gene and the non-structural protein 3C gene.
  • P1-2A upstream 5,-ATAGGATCCACCATGGGAGCCGGGCAATCCAGCC-3,
  • Total RNA was extracted from the foot-and-mouth disease virus cell culture medium.
  • the extracted total RNA was subjected to 0 0 ((11 18 primers under the action of AMV reverse transcriptase, reverse transcription at 42 ° C to prepare cDNA.
  • the obtained cDNA was used as a template, and PCR amplification was carried out with a specific antibody.
  • the PCR reaction system is as follows:
  • the PCR-amplified P1-2A and 3C gene products were subjected to 1% agarose gel electrophoresis, and it was found that a fragment of about 2.4 kb 0.7 kb was amplified.
  • the gel containing the corresponding DNA fragment was cut with a sterilized scalpel under a UV lamp and then purified using a Geneclean kit.
  • the method is as follows: Cut the gel fragment and weigh it, place it in a sterile 1.5 ml small centrifuge tube, add 3 times (v/w) volume of 6 M Nal, dissolve the gel at 37 °C, add 10 Glass milk, mix and leave at room temperature for 5 minutes, allow the DNA to fully adsorb on the glass milk, centrifuge at 12000 rpm for 5 seconds, then wash it three times with New Wash solution, each time the pellet is bounced and centrifuged. . Finally, the precipitate was air-dried, and 30 l of O.l xTE Buffer was added to dissolve the DNA. After centrifugation, the precipitate was removed, and the supernatant was taken for further analysis.
  • Digestion reaction Purified P1-2A was analyzed by double digestion with «7 HI and coRI. The total volume of the reaction was 50 ⁇ , of which 10 ⁇ of the purified PCR product and 5 ⁇ of the corresponding buffer of 10 ⁇ enzyme. 1 ⁇ 1, sterile water to make up Volume. The reaction was carried out at 37 ° C for 2 hours or more. The transfer plasmid pGEM-3Z was subjected to the same digestion reaction. After the reaction was completed, the mixture was inactivated at 65 ° C for 10 minutes.
  • Ligation reaction Total volume of ligation 15 ⁇ , PCR product 8 ⁇ l, vector 1 ⁇ , 5xT4 DNA ligation buffer 3 ⁇ l, ⁇ 4 DNA ligase 1 ⁇ , make up the volume with sterile water, and connect overnight at 12 ⁇ 14 °C.
  • the 3C, pGEM-3Z were digested by the same method, and the ligation reaction was carried out.
  • E. coli TG1 competent cells were prepared with 75 mM CaCl 2 . Take 5 ⁇ of the ligation mixture prepared in step 3, add to 200 ⁇ of competent cells, mix gently, bath for 30 minutes, heat at 42 °C for 2 minutes, quickly place on ice for 1-2 minutes, add warm Incubate to LB medium at 37 °C for 500 ⁇ , incubate at 37 °C for 1 hour, take 100-200 ⁇ , apply to LB solid medium plate containing 100 g/ml ampicillin (Amp), and incubate at 37 °C. overnight.
  • Amp ampicillin
  • the plasmid DNA prepared in 1.5 was double-digested with Bamm/EcoRI, and a plasmid having a DNA band of about 2.4 kb and 0.7 kb after electrophoresis was used as a recombinant carrying plasmid pGEM-3Z (P1-2A).
  • the plasmid DNA prepared in 1.5 was digested with EcoRI/BgiU, and the plasmid with 0.7 kb DNA band after electrophoresis was used as the recombinant carrying plasmid pGEM-3Z.
  • the recombinant plasmid pGEM-3Z (P1-2A pGEM-3Z (3C) was subjected to bidirectional sequencing.
  • the P1-2A3C gene sequence and amino acid sequence are shown in SEQ ID ⁇ : 1 and SEQ ID NO: 2, respectively.
  • the plasmid pGEM-3Z (P1-2A) prepared in 1.6 was digested with BamlU and EcoRI, and the P1-2A gene fragment was purified by the method of 1.2, and the baculovirus transfer vector PVL1393 double-digested with Bamm and coRI. After ligation, E. coli TG1 was transformed and the recombinant transfer vector pVL1393 (P1-2A) was selected.
  • the plasmid pGEM-3Z (3C) prepared in 1.6 was digested with EcoRI and Bglli, and the 3C gene fragment was purified by the method of 1.2, and ligated with the baculovirus transfer plasmid pVL1393 (P1-2A) double-digested with coRI and BglU. After transformation of E. coli TG1, the recombinant transfer vector pVL 1393 (P1-2A3C) was screened.
  • the silkworm cell BmN was cultured under C.
  • the silkworm nuclear polyhedrosis virus parent strain Bm-NPV-ZJ8 was used to infect about 50 ml of cells in the logarithmic growth phase, and the infection was collected for 1, 3 to 4 days.
  • the virus infection solution was collected and centrifuged (5000 rpm x lO min) to remove the precipitate.
  • virus DNA extract Tris 12.1 g, EDTA 33.6 g, KC1 14.1 g, pH 7.5 in 1000 ml
  • plaques containing no polyhedron were selected and the above procedure was repeated. After 2 to 3 rounds of purification, pure recombinant silkworm baculovirus rBmNPV (P1-2A3C) was obtained (the microbial deposit number is: CGMCC NO.1979).
  • the recombinant silkworm baculovirus rBmNPV (P1-2A3C) was infected with normal growing BmN cells, and the supernatant was collected after 3 days of culture. The supernatant contained a large amount of recombinant virus rBmNPV (P1-2A3C X
  • the integration of foreign genes was analyzed by PCR.
  • the extraction method of free viral genomic DNA is as follows: Take the virus supernatant 150 ⁇ 1, add 150 ⁇ 1 (0.5 mol/L) NaOH, mix well, add 20 ⁇ 1 (8 mol/L) ammonium acetate, mix the hook and use an equal volume of phenol. It was extracted once with chloroform, and after ethanol precipitation, the DNA was dissolved with 20 ⁇ l of TE.
  • Oligonucleotide primers are:
  • the high-expression variety of silkworm used in this experiment is JY1 (preserved by our laboratory).
  • the silkworm rearing of JY1 variety was carried out according to the conventional method of "Chinese Sericulture” edited by Lu Hongsheng (Shanghai Science and Technology Press, 1991).
  • the silkworms with the same average body weight were selected 48 h after foraging.
  • Each silkworm was inoculated with about 1.0 ⁇ 10 5 rBmNP (P1-2A3C). After 4-5 days, the hemolymph of the silkworm was collected and frozen at -20 V for double antibody sandwich ELISA. .
  • the 96-well microtiter plate was coated with rabbit anti-foot-and-mouth disease positive serum at 4 °C overnight.
  • the foot-and-mouth disease positive antigen, infection rBmNPV (P1-2A3C) Silkworm blood harvested from silkworm, silkworm blood infected with Bm-NPV-ZJ8, was diluted 2 times in silkworm. After 1 h at 37 °C, it was washed, and guinea pig anti-FMDV positive serum was added and treated at 37 °C for 1 h. After washing, HRP-rabbit anti-guinea pig IgG was added and treated at 37 °C for 1 h.
  • the substrate OPD-H 2 O 2 was added and allowed to act at 37 ° C for 15 min, and the reaction was terminated with a stop solution, and the OD value was measured at ⁇ 492 ⁇ .
  • Results As shown in Fig. 1, the hemolymph OD value obtained from the silkworm infected with rBmNPV (P1-2A3C) gradually decreased with the increase of the dilution ratio, and the change rule was the same as that of the positive control traditional cell vaccine virus antigen. From the results of the OD value measurement experiment, the expression level reached 100 times higher than that of the positive control traditional cell vaccine virus antigen, while no antigen expression was detected in the silkworm hemolymph of the control Bm-NPV-ZJ8 infection.
  • the silkworm cocoon used in this experiment is a highly expressed variety of JY1 (preserved by the laboratory).
  • the silkworm rearing of JY1 variety was carried out according to the conventional method of "Chinese Sericulture” edited by Lu Hongsheng (Shanghai Science and Technology Press, 1991). After seven days of crusting, 15 silkworm cocoons with the same average body weight were selected. The silkworms with the same average body weight were selected 48 h after the foraging. Each silkworm was inoculated with about l.Ox lO 5 rBmNPV (P1-2A3C), and the cocoon was collected 4-5 days later. Hemolymph, frozen at -20 °C for double antibody sandwich ELISA.
  • the hemolymph OD value obtained from the silkworm pupa infected with rBmNPV (P1-2A3C) gradually decreased with the increase of the dilution ratio > the change rule and the change of the OD value of the traditional cell vaccine virus antigen of the positive control The same rules. From the results of the OD value measurement experiment, the expression level reached 100 times higher than that of the positive control traditional cell vaccine virus antigen, while no antigen expression was detected in the hemolymph of the silkworm infected with the control Bm-NPV-ZJ8.
  • the Sephadex xerogel was weighed, swollen and placed in a glass column, and washed with an eluent (5 mmol/LTris solution, 0.1 mol/L NaCl, pH 8.0) until the baseline was stable.
  • the silkworm blood collected in the above 5 and 6 was ultrasonically disrupted and centrifuged to remove cell debris.
  • the above sample was loaded, and an appropriate amount of eluate was taken at a flow rate of 0.3 ml/min.
  • the protein eluate was collected at 5 min/tube, collected to the first peak, and the collected protein eluates were combined to obtain a purified foot-and-mouth disease antigen. .
  • the collected purified antigen is mixed with an equal volume of oil adjuvant. Take 3ml / head and neck muscles to immunize cattle. The real group was immunized with 5 cows and a control group of 2 cows was established. Before the vaccination, the antibody level of the candidate cattle was tested by the OIE standard method liquid phase blocking ELISA method, and the cattle whose titer was less than 1/8 were selected in Lanzhou. Experiment in the isolation animal room of the Veterinary Research Institute
  • Bovine serum was collected 21 days after immunization, and the anti-FMDV antibody levels in bovine serum were detected by liquid phase blocking ELISA. All the experimental animals achieved high antibody levels. Bovine tongue was inoculated with 10,000 BID 5() homologous virus for 10 days, and the incidence of the lips and four hooves was detected. Five animals in the experimental group were fully protected, while the control animals were all ill.
  • Example 2 Preparation, Purification, Animal Immunization Experiment and Virus Attack Protection Experiment of Foot-and-Mouth Disease Antigen
  • Primers were designed to amplify the full-length ORF of the foot-and-mouth disease virus by RT-PCR (the amplified primers of the amplified full-length ORF designed by SEQ ID ⁇ : 3 ⁇ are
  • ORF 5'-ATAGCGGCCGCAGGGATTATGCGTCACCGCACAC-3 '.
  • Total RNA was extracted from the foot-and-mouth disease virus cell culture medium.
  • the extracted total RNA was subjected to reverse transcription at 42 °C using a 0 0 ( ) 18 primer under the action of AMV reverse transcriptase to prepare cDNA.
  • the obtained cDNA was used as a template, and PCR amplification was carried out using specific primers.
  • the PCR reaction system is as follows:
  • PCR reaction process 94. C denatured for 10 minutes; 94. C 1 minute, 58 ° C 1 minute, 72. C 8 minutes, a total of 30 cycles. The reaction was finally extended for 10 minutes.
  • the ORF amplified in the above method was purified by the method of 1.2 in Example 1, and digested with Spel and NWI, and then ligated with the scorpion virus transfer vector pVL1393 digested with Xbal and ⁇ , and then transformed into Escherichia coli TG1.
  • Recombinant transfer vector pVL1393 ORF
  • the recombinant plasmid pVL1393 (ORF) was subjected to bidirectional sequencing.
  • the ORF gene sequence and amino acid sequence are shown in SEQ ID ⁇ : 3 and SEQ ID NO: 4, respectively.
  • plaques containing no polyhedron were selected and the above procedure was repeated. After 2 to 3 rounds of purification, pure recombinant silkworm baculovirus rBmNPV (ORJF) was obtained (the microbial deposit number is: CGMCC NO. 1980).
  • the recombinant silkworm baculovirus rBmNPV (ORF) was infected with normal growing BmN cells, and the supernatant was collected for 3 days, and the supernatant contained a large amount of recombinant virus rBmNPV (ORF).
  • the integration of foreign genes was analyzed by PCR.
  • the extraction method of free viral genomic DNA is as follows: Take the virus supernatant 150 ⁇ 1, add 150 ⁇ 1 (0.5 mol/L) NaOH, mix well, then add 20 ⁇ 1 (8 mol/L) Ammonium acetate, after mixing, was extracted once with an equal volume of phenol and chloroform, and after ethanol precipitation, the DNA was dissolved with 20 ⁇ l of hydrazine.
  • Oligonucleotide primers are:
  • the high-expression variety of silkworm used in this experiment is JY1 (preserved by our laboratory).
  • the silkworm rearing of JY1 variety was carried out according to the conventional method of "Chinese Sericulture” edited by Lu Hongsheng (Shanghai Science and Technology Press, 1991).
  • the silkworms with the same average body weight were selected 48 h after foraging.
  • Each silkworm was inoculated with l.Ox lO 5 rBmNPV (ORF). After 4-5 days, the hemolymph of the silkworm was collected and frozen at -20 ⁇ for double antibody sandwich ELISA.
  • the 96-well microtiter plate was coated with rabbit anti-FMDV positive serum for 4 overnight.
  • the FMDV antigen, the silkworm blood infected with the rBmNPV (ORF) infected silkworm, and the silkworm blood harvested from the silkworm infected with Bm-NPV-ZJ8 were diluted 2-fold. After being treated for 1 h at 37 °C, it was washed, and guinea pig anti-FMDV positive serum was added and treated at 37 °C for 1 h. After washing, HRP-rabbit anti-guinea pig IgG was added and treated at 37 °C for 1 h.
  • the substrate OPD-H 2 O 2 was added and allowed to act at 37 ° C for 15 min, and the reaction was terminated with a stop solution, and the OD value was measured at ⁇ 492 ⁇ .
  • Results As shown in Fig. 3, the hemolymph OD value obtained from the silkworm infected with rBmNPV (ORF) gradually decreased with the increase of the dilution ratio, and the change rule was the same as that of the positive control traditional cell vaccine virus antigen. From the results of the OD value measurement experiment, the expression level reached 10 times higher than that of the positive control traditional cell vaccine virus antigen, while no antigen expression was detected in the hemolymph of the silkworm infected with the control Bm-NPV-ZJ8.
  • the silkworm cocoon used in this experiment is a highly expressed variety of JY1 (preserved by the laboratory).
  • the silkworm rearing of JY1 variety was carried out according to the conventional method of "Chinese Sericulture” edited by Lu Hongsheng (Shanghai Science and Technology Press, 1991). After the crusting for seven days, 15 silkworm pupa with the same average body weight were selected. The cocoon with the same average body weight was selected 48 h after the foraging.
  • Each silkworm cocoon was inoculated with about 1.0 ⁇ 10 5 pfu rBmNPV (ORP), and the silkworm sputum hemolymph was collected after 4-5 days. °C was frozen for double antibody sandwich ELISA. Results As shown in Fig.
  • the Sephadex xerogel was weighed, swelled and placed on a glass column, and washed with an eluent (5 mmol/LTris solution, 0.1 mol/L NaCl, pH 8.0) until the baseline was stable.
  • the silkworm blood lymphocytes harvested in 4 and 5 were broken by ultrasonic waves and centrifuged to remove cell debris.
  • the above sample was loaded, and an appropriate amount of eluate was taken at a flow rate of 0.3 ml/min.
  • the protein eluate was collected at 5 min/tube, collected to the first peak, and the protein eluate was combined to obtain a purified antigen.
  • Bovine serum was collected 21 days after immunization, and the anti-FMDV antibody levels in bovine serum were detected by liquid phase blocking ELISA. All the experimental animals achieved high antibody levels. ⁇ Inoculation of beef tongue with 10,000 BID 5 () homologous virus, continuous observation for 10 days, detection of the incidence of the lips and four hooves, the experimental group of 5 animals obtained 4 protection, while the control group of all the disease.
  • Example 3 Preparation, Purification, Animal Immunization Experiment and Virus Attack Protection Experiment of Foot-and-Mouth Disease Antigen
  • Primers were designed to amplify the foot-and-mouth disease virus VP 1 gene by RT-PCR.
  • the designed amplification primer for the amplified VP 1 gene is
  • VP1 upstream 5 '-ATAGGATCCACCATGGCCACCACTACCGGCGAGTCAG-3 ',
  • the PCR reaction system is as follows: Table 3 PCR reaction conditions
  • PCR reaction process 94. C denaturation for 10 minutes; 94 °C for 1 minute, 58 °C for 1 minute, and 72 °C for 1 minute for a total of 30 cycles. Finally, the reaction was extended for 10 minutes.
  • the obtained VP1 gene fragment was purified by the method of 1.2 in Example 1, and digested with ⁇ and EcoRI, and then ligated with the same double-cut baculovirus transfer vector pVL1393, and then transformed into Escherichia coli TG1. Transfer the pVL1393 (VP1).
  • the recombinant plasmid pVL1393 (VP1) was subjected to bidirectional sequencing.
  • the VP1 gene sequence and amino acid sequence are shown in SEQ ID NO: 5 and SEQ ID NO: 6, respectively.
  • the infection solution was aspirated, and the 2% low melting point agarose gel was melted in a 60 °C water bath, and cooled to 40 °C and 40 °C preheated 2xTC-100 medium (including 20 % FBS ) Mix well, add 4 ml of glue per level. After solidification, seal with Parafilm, incubate at 27 °C for 3 to 5 days, and observe under microscope. The plaques containing no polyhedron were selected and the above procedure was repeated. After 2 to 3 rounds of purification, pure recombinant silkworm baculovirus rBm PV (VP1) was obtained (the microbial deposit number is: CGMCC NO.1975).
  • the recombinant silkworm baculovirus rBmNPV (VP1) was infected with normal growth BmN cells, and the supernatant was collected after 3 days of culture. The supernatant contained a large amount of recombinant virus rBmNPV (VP1).
  • the integration of foreign genes was analyzed by PCR.
  • the extraction method of free viral genomic DNA is as follows: Take the virus supernatant 150 ⁇ 1, add 150 ⁇ 1 (0.5 mol/L) NaOH, mix well, add 20 ⁇ 1 (8 mol/L) ammonium acetate, mix the hook and use an equal volume of phenol. It was extracted once with chloroform, and after ethanol precipitation, the DNA was dissolved with 20 ⁇ l of TE.
  • Oligonucleotide primers are:
  • reaction conditions are: 94 °C denaturation 5 min, 94 °C lmin, 55 °C lmin, 72 °C lmin, 30 cycles, last 72 °C extended for 5 min.
  • the 15 ⁇ l reaction product was subjected to electrophoresis analysis, and it was confirmed that the recombinant virus was obtained.
  • the high-expression variety of silkworm used in this experiment is JY1 (preserved by our laboratory).
  • the silkworm rearing of JY1 variety was carried out according to the conventional method of "Chinese Sericulture” edited by Lu Hongsheng (Shanghai Science and Technology Press, 1991).
  • the silkworms with the same average body weight were selected 48 h after foraging.
  • Each silkworm was inoculated with about 1.0 ⁇ 10 5 rBmNPV (VP1). After 4-5 days, the hemolymph of the silkworm was collected and frozen at -20 °C for double antibody sandwich ELISA. Results As shown in Fig.
  • the silkworm cocoon used in this experiment is a highly expressed variety of JY1 (preserved by the laboratory).
  • the silkworm rearing of JY1 variety was carried out according to the conventional method of "Chinese Sericulture” edited by Lu Hongsheng (Shanghai Science and Technology Press, 1991). After seven days of crusting, 15 silkworm pupa with the same average body weight were selected. The cocoon with the same average body weight was selected 48 h after the foraging.
  • Each silkworm cocoon was inoculated with about 1.0 ⁇ 10 5 rBm PV (VP1), and the silkworm hemolymph was collected after 4-5 days. 20 ⁇ frozen for detection by double antibody sandwich ELISA. Results As shown in Fig.
  • the hemolymph OD value obtained from the silkworm infected with rBm PV (ORF) gradually decreased with the increase of the dilution ratio, and the change rule was the same as that of the positive control traditional cell vaccine virus antigen. From the results of the OD value measurement experiment, the expression level reached 100 times higher than that of the positive control traditional cell vaccine virus antigen, while no antigen expression was detected in the silkworm hemolymph of the control Bm-PV-ZJ8 infection.
  • the Sephadex xerogel was weighed, swelled and placed in a glass column, and washed with an eluent (5 mmol LTris solution, 0.1 mol/L NaCl, pH 8.0) until the baseline was stable.
  • the silkworm blood lymphocytes harvested in 4 and 5 were broken by ultrasonic waves and centrifuged to remove cell debris.
  • the above sample was loaded, and an appropriate amount of eluate was taken at a flow rate of 0.3 ml/min.
  • the protein eluate was collected at 5 min/tube, collected to the first peak, and the protein eluate was combined to obtain a purified antigen.
  • the silkworm blood lymphocytes collected in 4 or 5 were ultrasonically disrupted, and the cell debris was discarded and mixed with an equal volume of oil adjuvant. Take 3ml/head and neck muscles to immunize cattle.
  • the experimental group was immunized with 5 cows and a control group of 2 cows was established. Before the vaccination, the antibody was tested by the OIE standard method liquid phase blocking ELISA method, and the cattle whose titer was less than 1/8 were selected for experiment in the isolation animal room of Lanzhou Veterinary Research Institute.
  • Bovine serum was collected 21 days after immunization, and the level of anti-foot-and-mouth disease antibody in bovine serum was detected by liquid phase blocking ELISA. All the experimental animals achieved high antibody levels.
  • the tongue was inoculated with a 10,000 BID 50 homologous virus for 10 days, and the incidence of the lips and the four hooves was detected. Four of the five animals in the experimental group were protected, while the control animals were all ill.

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Abstract

L'invention porte sur une méthode d'expression d'antigènes de virus de la fièvre aphteuse dans l'insecte in vivo en utilisant des baculovirus de recombinaison comprenant les étapes suivantes: clonage de différentes combinaison de gènes du virus de la fièvre aphteuse dans des vecteurs portant le baculovirus pour construire le vecteur de transfert; transfection du baculovirus avec le vecteur de transfert pour obtenir la recombinaison de l'ADN et le baculovirus de recombinaison; infection de l'insecte hôte par le baculovirus de recombinaison; culture de l'insecte hôte infecté pour exprimer des antigènes du virus de la fièvre aphteuse; et recueil et purification des antigènes exprimés. Les gènes préférés porteurs des vecteurs du baculovirus, du baculovirus et l'insecte hôte sont choisis parmi les gènes P1-2A3C, ORF et VP1 du virus de la fièvre aphteuse se trouvant dans SEQ ID No 1, 3 ou 5, pVL1393, Bombyx mori Bm-NPV-ZJ8, et des larves ou nymphes du Bombyx mori.
PCT/CN2008/000126 2007-03-23 2008-01-17 Méthode de préparation d'antigènes du virus de la fièvre aphteuse WO2008116368A1 (fr)

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JP2013509203A (ja) * 2009-11-02 2013-03-14 ザ トラスティーズ オブ ザ ユニバーシティ オブ ペンシルバニア 口蹄疫ウイルス（ｆｍｄｖ）コンセンサスタンパク質、そのコード配列およびそれから生成されるワクチン
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JP2013509203A (ja) * 2009-11-02 2013-03-14 ザ トラスティーズ オブ ザ ユニバーシティ オブ ペンシルバニア 口蹄疫ウイルス（ｆｍｄｖ）コンセンサスタンパク質、そのコード配列およびそれから生成されるワクチン
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US9109014B2 (en) 2009-11-02 2015-08-18 The Trustees Of The University Of Pennsylvania Foot and mouth disease virus (FMDV) consensus proteins, coding sequences therefor and vaccines made therefrom
JP2015166353A (ja) * 2009-11-02 2015-09-24 ザ トラスティーズ オブ ザ ユニバーシティ オブ ペンシルバニア 口蹄疫ウイルス（ｆｍｄｖ）コンセンサスタンパク質、そのコード配列およびそれから生成されるワクチン
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US11124545B2 (en) 2009-11-02 2021-09-21 The Trustees Of The University Of Pennsylvania Foot and mouth disease virus (FMDV) consensus proteins, coding sequences therefor and vaccines made therefrom
US12049479B2 (en) 2009-11-02 2024-07-30 The Trustees Of The University Of Pennsylvania Foot and mouth disease virus (FMDV) consensus proteins, coding sequences therefor and vaccines made therefrom
CN101816297A (zh) * 2010-04-30 2010-09-01 中国计量学院 一种诱捕防治茶尺蠖成虫的方法
US9993545B2 (en) 2013-03-15 2018-06-12 The Trustees Of The University Of Pennsylvania Foot and mouth disease virus (FMDV) consensus proteins, coding sequences therefor and vaccines made therefrom

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