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H7N9 subtype avian influenza genetic engineering vaccine taking baculovirus as carrier as well as preparation method and application of vaccine

A genetically engineered vaccine and baculovirus technology, applied in the field of H7N9 subtype avian influenza genetically engineered vaccine and its preparation, can solve the problems of chicken embryo production limitation, hidden biological safety hazards, antigen pollution, etc., and achieve the convenience of large-scale production, Facilitate mass production and improve the expression level effect

Inactive Publication Date: 2017-02-22
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The antigenicity of the H7N9 subtype avian influenza virus is different from that of the previous avian influenza viruses. The original seasonal influenza vaccine and avian influenza vaccine lack immune protection against it. The most widely used oil emulsion inactivated vaccine is the traditional one. Although oil emulsion inactivated vaccines can induce sufficient immune protection in immunized animals, there are also some defects: the production of vaccines is overly dependent on chicken embryos, and the output of chicken embryos is limited during influenza outbreaks; there is the possibility of other antigen contamination; inactivation The anti-NP or M1 protein antibodies produced after vaccine immunization cannot distinguish the antibodies produced by immunization from wild virus infection
However, the attenuated live vaccine is infectious and has biological safety hazards, so it is not allowed to be used in poultry at present

Method used

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  • H7N9 subtype avian influenza genetic engineering vaccine taking baculovirus as carrier as well as preparation method and application of vaccine
  • H7N9 subtype avian influenza genetic engineering vaccine taking baculovirus as carrier as well as preparation method and application of vaccine
  • H7N9 subtype avian influenza genetic engineering vaccine taking baculovirus as carrier as well as preparation method and application of vaccine

Examples

Experimental program
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Effect test

Embodiment 1

[0028] Embodiment 1: pFast-(CMV) in the present invention 2 Construction of Backbone Plasmids

[0029] (1) Construction and identification of recombinant plasmid pFast-CMV

[0030] Using the pcDNA3.1(+) plasmid as a template, two pairs of primers were designed, P1, P2 and P3, P4. Among them, primers P1 and P2 were used to amplify the human cytomegalovirus CMV promoter, and BstZ 17I and BbsI restriction sites were introduced upstream and downstream, and the primer sequences were as follows:

[0031] P1: 5'-GTATACGTTGACATTGATTATTGACTAGTTATTAATAGTAATCAATT-3'

[0032] P2: 5'-GAAGACTTGATCACACAGCTTGGGTCTCCCTATAGTGAGT-3'

[0033] The amplification conditions of primers P1 / P2 were: pre-denatured at 94°C for 5 minutes and then cycled; cycle parameters were: 94°C, 45s, 56°C, 45s, 72°C, 1min; after 30 cycles, final extension at 72°C for 10 minutes. The PCR product was recovered and digested with BstZ 17I and BbsI, and then inserted into the vector pFastBac TM Dual's BstZ 17I and Bbs...

Embodiment 2

[0038] Embodiment 2: Construction and identification of recombinant bacmid (Bacmid)

[0039] (1) Extraction of H7N9 subtype avian influenza virus genome RNA:

[0040] The H7 subtype avian influenza virus A / CK / GD / G1 / 2013 (H7N9) (hereinafter referred to as G1) used in this experiment and the positive serum prepared from it were all provided by the National Center for Prevention and Control of Zoonotic Diseases of South China Agricultural University. Saved by Joint Engineering Laboratories. The chicken embryo allantoic fluid containing G1 virus was centrifuged at 12,000 r / min for 5 min, and 500 μL of the supernatant was taken into a DEPC-treated Eppendorf tube to extract RNA.

[0041] ① Add 500 μL Trizol LS Reagent, shake vigorously and place at room temperature for 10 minutes, add 200 μL chloroform, mix thoroughly, wait for the emulsified solution to become milky, leave it at room temperature for 10 minutes, and then centrifuge (13000rpm, 15min, 4°C);

[0042] ② Transfer 600 μ...

Embodiment 3

[0059] Embodiment 3: recombinant baculovirus BV-(CHA) 2 acquisition and identification of

[0060] (1) Recombinant baculovirus BV-(CHA) 2 the acquisition

[0061] Using liposome-mediated transfection, the recombinant bacmid-(CHA) 2 Transfect sf9 insect cells and culture at 27°C. When the cells were cultured for 72 hours, lesions appeared in the cells, and the cell culture supernatant was collected to obtain the first-generation recombinant baculovirus (P1)BV-(CHA) 2 .

[0062] (2) Recombinant baculovirus BV-(CHA) 2 Transduction of PK15 cells and identification of its expression ability

[0063] 1 day before transduction, the well-formed PK15 cells were divided into 1 × 10 5 The amount of each well was inserted into a 6-well cell culture plate, and cultured in a 37°C incubator. After the cells grew to 70%-80% monolayer, the medium was removed, and the cells were washed with PBS containing calcium and magnesium ions. After mixing the recombinant baculovirus and PBS at a ra...

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Abstract

The invention discloses an H7N9 subtype avian influenza genetic engineering vaccine taking baculovirus as a carrier as well as a preparation method and an application of the vaccine and belongs to the technical field of genetic engineering. The H7N9 subtype avian influenza genetic engineering vaccine takes the baculovirus as the carrier and is a recombinant baculovirus capable of expressing main protective antigens of the avian influenza virus in vertebrate. The avian influenza genetic engineering vaccine realizing immunity through intramuscular injection is prepared. The vaccine overcomes defects that inactivated vaccines depend on chicken embryo production, the production period is long, other antigen mixed infection exists and the like, and technical reserve is provided for screening the H7N9 subtype avian influenza genetic engineering vaccine.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a H7N9 subtype avian influenza genetic engineering vaccine with a baculovirus as a carrier, a preparation method and application thereof. Background technique [0002] Avian influenza virus (AIV) belongs to the Orthomyxoviridae Influenza virus genus, which has always caused serious harm to the breeding industry and human public safety. The outbreak of H7N9 subtype AIV in 2013 has aroused global attention to AIV. Although H7N9AIV has low pathogenicity to poultry and generally does not show clinical symptoms after infection, it is in a virus-carrying state, but it has strong pathogenicity to humans. In order to effectively control poultry infection of this subtype of AIV, it is urgent to develop a vaccine that can effectively prevent avian H7N9 influenza virus. [0003] Immunization of susceptible animals with appropriate vaccines can protect susceptible b...

Claims

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Application Information

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IPC IPC(8): A61K39/145A61P31/16C12N15/866C12N15/44
CPCA61K39/12A61K2039/5256A61K2039/53C07K14/005C12N15/86C12N2710/14043C12N2760/16122C12N2760/16134
Inventor 廖明张娇樊惠英章震
Owner SOUTH CHINA AGRI UNIV
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