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Recombinant H9N2 avian influenza virus strain, preparation method thereof, avian influenza vaccine and application thereof

A technology of avian influenza virus and recombinant virus, applied in botany equipment and methods, biochemical equipment and methods, applications, etc., can solve the problems of high early mortality and final mortality of chicken embryos, increasing injection doses, affecting vaccine application, etc. , achieving significant economic benefits, increased replication titers, and large market applicability

Active Publication Date: 2019-06-14
YANGZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in the production process, there are problems such as high early death rate of chicken embryos and high final mortality rate, two small harvests per embryo on average, and unstable titer.
In order for the immunized animals to obtain a sufficient amount of avian influenza virus antigen, it is necessary to increase the injection dose, thereby affecting the application of the vaccine

Method used

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  • Recombinant H9N2 avian influenza virus strain, preparation method thereof, avian influenza vaccine and application thereof
  • Recombinant H9N2 avian influenza virus strain, preparation method thereof, avian influenza vaccine and application thereof
  • Recombinant H9N2 avian influenza virus strain, preparation method thereof, avian influenza vaccine and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Embodiment 1: Extraction of F / 98 strain RNA

[0037] F / 98 strain was inoculated in SPF chicken embryos, collected allantoic fluid 300ml, centrifuged at 6000rpm (30# rotor) for 15min, and took the supernatant; centrifuged at 18000rpm at 4°C for 1.5h, washed with 40ml STE (10mM pH8.0Tris-HCl, 100mM NaCl, 5mM pH8.0EDTA) to suspend the precipitate. Use 10% sucrose as the base, carefully add the suspension, centrifuge at 18000rpm 4°C for 1.5h to remove impurities, discard the supernatant, suspend the precipitate with 30mL STE, centrifuge at 18000rpm 4°C for 1h to remove sucrose, suspend the precipitate with 7mL STE, and pack into finger tubes , 450 μL per tube.

[0038] RNA was extracted with the RNA extraction kit from Shanghai Bioengineering Technology Service Co., Ltd. Take 300 μL of viral allantoic fluid, add 400 μL Trizol and mix thoroughly; add 100 μL chloroform / isoamyl alcohol (24:1), shake for 30 seconds, and centrifuge at 12,000 rpm for 5 minutes; transfer the sup...

Embodiment 2

[0039] Embodiment 2 Amplification, cloning and sequence determination of 8 genes of F / 98 strain

[0040] Primers were designed to amplify the 8 gene sequences of F / 98 strain and sequenced.

[0041] The primer sequences for amplifying the 8 internal genes of the F / 98 strain are as follows:

[0042] Bm-HA-1 5'-TATTCGTCTCAGGGAGCAAAAGCAGGGG-3',

[0043] Bm-HA-2 5-ATATCGTCTCGTATTAGTAGAAACAAGGGTGTTTT-3'.

[0044] Bm-NA-1 5'-TATTCGTCTCAGGGAGCAAAAGCAGGAGT-3';

[0045] Bm-NA-2 5'-ATATCGTCTCGTATTAGTAGAAACAAGGAGTTTTTT-3',

[0046] Bm-PB1-1 5'-TATTCGTCTCAGGGAGCGAAAGCAGGCA-3';

[0047] Bm-PB1-2 5'-ATATCGTCTCGTATTAGTAGAAACAAGGCATTT-3',

[0048] Bm-PA-1 5'-TATTCGTCTCAGGGAGCGAAAGCAGGTACTGAT-3';

[0049] Bm-PA-2 5'-ATATCGTCTCGTATTAGTAGAAACAAGGTACTTTT-3',

[0050] Bm-NP-1 5'-TATTCGTCTCAGGGAGCAAAAGCAGGGTAGATAATC-3';

[0051] Bm-NP-2 5'-ATATCGTCTCGTATTAGTAGAAACAAGGGTATTTTTC-3';

[0052] Bm-PB2-1 5'-TATTCGTCTCAGGGAGCGAAAGCAGGTC-3';

[0053] Bm-PB2-2 5'-ATATCGTCTCGTATTAGTAGAAACAAGGTCGTTT-...

Embodiment 3

[0071] Embodiment 3 constructs the transcription / expression vector plasmid of 8 genes of F / 98 strain:

[0072] In the 8-plasmid transcription / expression vector pHW2000, insert the cDNA of 8 internal genes (PB2, PB1, PA, HA, NP, M, NS, NA) of the F / 98 strain to form 8 reassortant plasmids: pHW202-PB2, pHW202 - PB1, pHW203-PA, pHW204-HA, pHW205-NP, pHW206-NA, pHW207-M ​​and pHW208-NS.

[0073] After sequence analysis, the correct sequence was selected for cloning into the 8-plasmid transcription / expression vector pHW2000. First, 6 internal genes of F / 98 strain, NA gene of D3 strain and 8 plasmid transcription / expression vector pHW2000 were digested with BsmBI. The BsmBI digestion system (40 μL) was as follows, and 5 μL of plasmid DNA (0.3 μg / μL) was taken. 55 ℃ for 4h.

[0074]

[0075] The enzyme-digested fragments and the vector were recovered by gel electrophoresis and ligated with T4 DNA ligase, transformed into Escherichia coli JM109 competent cells, picked colonies to...

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Abstract

The invention discloses a PB2 mutant gene, a mutant protein, a recombinant vector and a recombinant cell. The invention also discloses a recombinant virus and a preparation method and application thereof. The invention also discloses an H9N2 avian influenza vaccine and application thereof. 1185K and R355K amino acid mutations are respectively introduced to a PB2 gene of the H9N2 subtype avian influenza virus F / 98 strain, which can significantly enhance the replication capability of the F / 98 strain in a chicken embryo. The virus replication titer is significantly increased, and an effective technical method is provided for improving the efficiency of vaccine production and reducing the production cost. Great market applicability is achieved, and significant economic benefits are created.

Description

technical field [0001] The invention belongs to the field of vaccine production, and in particular relates to a recombinant H9N2 avian influenza virus strain, a preparation method thereof, an avian influenza vaccine and applications thereof. Background technique [0002] Avian influenza is a highly contagious infectious disease of poultry and wild poultry caused by type A influenza virus, which can be divided into high pathogenicity and low pathogenicity. The H9N2 subtype is a low-pathogenic influenza virus, which can cause a serious drop in egg production rate of poultry, and easily cause concurrent infection, resulting in serious economic losses. Since the outbreak of H9N2 influenza virus epidemic in my country in 1998, vaccines have played an important role in the prevention and control of the disease, especially the immune effect of avian influenza embryotoxin inactivated oil emulsion vaccine is extremely significant. The inactivated vaccine produced with H9N2 subtype a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/44C12N7/01C07K14/11A61K39/145A61P31/16
Inventor 石火英苏海龙
Owner YANGZHOU UNIV
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