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Preparation method of recombinant baculovirus vaccine for preventing and treating avian influenza H5N1

An avian influenza virus and virus vaccine technology, which is applied in the field of recombinant baculovirus vaccine preparation, can solve the problems of low efficiency and gene expression level, short duration of continuous expression of foreign genes, and limited application of baculovirus.

Inactive Publication Date: 2016-07-13
HEILONGJIANG UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, due to the low efficiency and gene expression level of baculovirus in transducing mammalian cells, and the short duration of foreign gene expression, the application of baculovirus as a poultry vaccine is limited. Therefore, how to improve the transduction of baculovirus Efficiency, exogenous gene expression efficiency and prolonging the expression time of exogenous genes have become research hotspots

Method used

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  • Preparation method of recombinant baculovirus vaccine for preventing and treating avian influenza H5N1
  • Preparation method of recombinant baculovirus vaccine for preventing and treating avian influenza H5N1
  • Preparation method of recombinant baculovirus vaccine for preventing and treating avian influenza H5N1

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Cloning of embodiment 1.HA gene

[0038] Lymphocytes were isolated from chicken peripheral blood, cultured for 8 hours, genomic RNA of chicken lymphocytes was extracted using TrizolReagent kit, the first strand of cDNA was obtained by reverse transcription, chicken HA gene was cloned by PCR, and it was connected to pMD18-T Vector, after verification and correct sequencing, named "pT-HA".

[0039] 1.1 Culture of Chicken Peripheral Blood Lymphocytes

[0040]Use a pipette to add 2 mL of lymphocyte separation solution to the bottom of a 15 mL centrifuge tube, avoiding contact with the side wall. Anticoagulant needles Take 3mL of chicken wing underarm blood, add 3mL LD-Hanks solution, mix with a pipette, pay attention to avoid air bubbles, slowly add to the lymphocyte separation medium, 2000r / min, after centrifugation for 20min, use 1mL Aspirate the buffy coat with the syringe, draw as little separation liquid as possible, and place it in a 15mL centrifuge tube. Add 5mL...

Embodiment 2

[0057] Construction of embodiment 2.pT-HA plasmid

[0058] After the bands were detected correctly, the remaining PCR products were recovered and purified, and the specific operation steps were performed according to the manual of GelExtractionMiniKit (Tiangen, M2029). Add "A" to the HA fragments recovered from the gel, the reaction system is shown in Table 6, and the reaction program settings are shown in Table 7. The recovered product after treatment was connected to the pMD18-T vector overnight at 16°C. The reaction system is shown in Table 8. The correct positive plasmid was named "pT-HA".

[0059] Table 6 PCR amplification product HA end plus "A" reaction system

[0060]

[0061] Table 7 PCR amplification product HA plus "A" reaction program

[0062]

[0063] Table 8 HA fragment and pMD18-T carrier connection reaction system

[0064]

Embodiment 3

[0065] Construction of embodiment 3.pS-ITRs-HA plasmid

[0066] The pS-ITRs-HA plasmid contains CMV promoter and regulatory elements such as WPRE, VSV-GED, ITRs and gp64sp, which were respectively obtained from the template plasmid pEGFP-C3 (purchased from Clontech Company), pRRLSIN. Addgene), pCMV-VSVG (purchased from addgene), pAAV-LacZ (purchased from Agilent) and Bacmid were cloned and identified.

[0067] 3.1 Cloning of each component

[0068] Using plasmids pEGFP-C3, pRRLSIN.cPPT.PGK-GFP.WPRE, pCMV-VSVG, pAAV-LacZ and Bacmid as templates, CMV-up, CMV-down, WPRE-up, WPRE-down, VSV-GED-up , VSV-GED-down, ITRs-L-up, ITRs-L-down, ITRs-R-up, ITRs-R-down and gp64sp-up, gp64sp-down as upstream and downstream primers, PCR amplification of CMV, WPRE, For genes of regulatory elements such as VSV-GED, ITRs, and gp64sp, the reaction system is shown in Table 4, and the reaction program settings are shown in Table 5. The results of PCR amplification were as figure 2 , image 3...

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Abstract

The invention relates to a preparation method of a recombinant baculovirus vaccine for preventing and treating avian influenza H5N1, in particular to an H5N1 virus vaccine strain for expressing avian influenza virus hemagglutinin HA gene and a preparation method thereof. The preparation method concretely comprises the following steps of 1) building pS-ITRs-HA plasmids; 2) converting the pS-ITRs-HA plasmids into E .coli DH10 Bac; 3) transfecting Sf9 insect cells by recombinant baculovirus shuttle vectors; 4) amplifying the recombinant baculovirus. When the recombinant H5N1 virus vaccine strain obtained through preparation is used for performing immunodetection on blood serum of immune chicken; the level of IgG antibodies in the blood serum of the immune chicken can reach 0.966 to the highest degree; the concentrations of IL-2, IL-4 and IFN-gamma can respectively reach 89 .67ng / L, 80 .21ng / L and 64 .24ng / L to the highest degree. The results show that the recombinant baculovirus vaccine can effectively irritate the chicken bodies to simultaneously generate the body fluid immunity and the cell immunity; the dual immunity protection effect is provided for the chicken group; the invention lays the theoretical foundation for the research and development of the avian influenza virus based on the baculovirus.

Description

technical field [0001] The invention relates to a preparation method of a vaccine, in particular to a preparation method of a recombinant baculovirus vaccine of avian influenza H5N1. Background technique [0002] The threat of avian influenza virus (Avian Influenza Virus) to poultry and humans is increasing day by day, and the prevention and control of avian influenza is becoming more and more important. At present, the prevention and treatment of avian influenza is mainly vaccine immunization. However, due to the large number of serotypes of H5N1 subtype highly pathogenic avian influenza and antigenic variation, traditional vaccines cannot provide strong protection; Long, high preparation cost, and safety constraints, it has not been widely used. Baculovirus is widely used in the transduction of insect cells and mammalian cells due to its advantages of no replication in host cells, high safety, and high protein expression. In the eukaryotic expression system, the baculovi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/01C12N15/866A61K39/145A61P31/16C12R1/93
CPCA61K39/12C07K14/005C12N7/00C12N15/86C12N2710/14021C12N2710/14043C12N2760/16122C12N2760/16134C12N2800/107C12N2800/60C12N2840/105C12N2840/60
Inventor 葛菁萍平文祥高冬妮安琪刘颖白程乐于航
Owner HEILONGJIANG UNIV
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