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Recombinant h9n2 avian influenza virus strain, preparation method thereof, avian influenza vaccine and application thereof

An avian influenza virus and recombinant virus technology, applied in the directions of botanical equipment and methods, biochemical equipment and methods, and applications, can solve the problems of high premature mortality and final mortality of chicken embryos, increasing the injection dose, and affecting the application of vaccines. , to achieve significant economic benefits, increased replication titer, and large market applicability.

Active Publication Date: 2022-03-25
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] Example 1: Extraction of F / 98 strain RNA

[0037] Strain F / 98 was inoculated into SPF chicken embryos, collected 300 ml of allantoic fluid, centrifuged at 6000 rpm (30# rotor) for 15 min, and took the supernatant; centrifuged at 18,000 rpm at 4°C for 1.5 h, and used 40 ml of STE (10 mM pH8.0 Tris-HCl, 100 mM NaCl, 5mM pH8.0 EDTA) to resuspend the pellet. Bottom with 10% sucrose, add the suspension carefully, centrifuge at 18000rpm for 1.5h at 4°C to remove impurities, discard the supernatant, suspend the pellet with 30mL STE, centrifuge at 18000rpm for 1h at 4°C to remove the sucrose, suspend the pellet with 7mL STE, and dispense into finger tubes , 450 μL per tube.

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

Embodiment 2

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

[0040] Primers were designed to amplify 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’-TATTCGTCTCAGGGAGCA AAAGCAGGGTAGATAATC-3’;

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

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

[0053] Bm-PB2-2 5’-ATATCGTCTCGTATTAGTAGAAACAAGGTCGTTT-3...

Embodiment 3

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

[0072] In the 8 plasmid transcription / expression vector pHW2000, 8 internal gene (PB2, PB1, PA, HA, NP, M, NS, NA) cDNAs of F / 98 strain were inserted into 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. Firstly, 6 internal genes of F / 98 strain, NA gene of D3 strain and 8 plasmid transcription / expression vector pHW2000 were digested with BsmBI enzyme. 55 ℃ role for 4h.

[0074]

[0075] The digested fragment and the vector were respectively subjected to gel electrophoresis, recovered, and then ligated with T4 DNA ligase, transformed into E. coli JM109 competent cells, picked for colony expansion and cultured, and a small amount of plasmid was extracted. ...

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Abstract

The invention discloses a PB2 mutant gene, mutant protein, recombinant vector and recombinant cell. The invention also discloses a recombinant virus and its preparation method and application. The invention also discloses a H9N2 bird flu virus vaccine and its application. The present invention respectively introduces 1185K and R355K amino acid mutations into the PB2 gene of the H9N2 subtype avian influenza virus F / 98 strain, so that the replication ability of the F / 98 strain in chicken embryos can be significantly enhanced, and the virus replication titer can be significantly increased. Improving vaccine production efficiency and reducing production cost provides an effective technical method. The invention has great market applicability and creates remarkable economic benefits.

Description

technical field [0001] The invention belongs to the field of vaccine production, and particularly 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 birds caused by influenza A virus, which is divided into high pathogenicity and low pathogenicity. The H9N2 subtype is a low pathogenic influenza virus, which can cause a serious decline in the egg production rate of poultry, and is prone to concurrent infection, resulting in serious economic losses. Since the outbreak of H9N2 influenza virus 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 embryovirus inactivated oil emulsion vaccine is extremely significant. The inactivated vaccine produced with H9N2 subtype avian influenza ...

Claims

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Application Information

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