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H5 subtype HA (hemagglutinin) expressing replication-defective recombinant H9N2 avian influenza virus

An avian influenza virus, replication-deficient technology, applied in the field of influenza virus vaccine technology research and development, can solve the problems of no attenuated live vaccine, virulence reversion mutation, etc., achieve strong and lasting immunogenicity, maintain immunogenicity, and good gene stability sexual effect

Inactive Publication Date: 2019-01-04
QINGDAO AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, vaccine immunization is still the most important and effective measure to prevent and control avian influenza. Only inactivated vaccines have been approved for use against H5N1 and H9N2 subtypes of avian influenza viruses, and there is no vaccine that can protect H9N2 and H5N1 subtypes of avian influenza at the same time. The attenuated live vaccine of influenza virus, and the traditional attenuated live vaccine of influenza virus is mainly a cold-adapted strain, which has the risk of virulence reverting mutation

Method used

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  • H5 subtype HA (hemagglutinin) expressing replication-defective recombinant H9N2 avian influenza virus
  • H5 subtype HA (hemagglutinin) expressing replication-defective recombinant H9N2 avian influenza virus
  • H5 subtype HA (hemagglutinin) expressing replication-defective recombinant H9N2 avian influenza virus

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Experimental program
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Embodiment 1

[0023] Embodiment 1 is used for synthesizing type A influenza virus NA ps -HA-NA ps DNA fragments and plasmid construction

[0024] Plasmids pHW-PB2, pHW-PB1, pHW-PA, pHW-HA, pHW-NP, pHW-M, pHW-NS containing seven segments of influenza A virus H9N2 were used. The above seven plasmids constitute a reverse genetic operating system with the backbone of influenza A virus H9N2.

[0025] 1. Construction for the synthesis of influenza A virus NA ps -HA-NA ps DNA fragment

[0026] Synthetic DNA sequence of type A influenza virus NA-HA (ORF): use the open reading frame (Open Reading Frame, ORF) of hemagglutinin HA of type A influenza virus A / wild duck / Hunan / 021 / 2005 (H5N1) subtype ) to replace the open reading frame of neuraminidase NA in influenza A virus A / Chicken / Shandong / 830 / 2014 (H9N2), while retaining the packaging signal region (203 base pairs) at the 5' end of the NA and the 3' end Packaging signal region (195 base pairs), synthesized a new DNA sequence named NA ps -HA(O...

Embodiment 2

[0033] Embodiment 2 rescues recombinant virus

[0034] Construction of cell lines: construction of MDCK cell lines that can stably express A / Chicken / Shandong / 830 / 2014 (H9N2) influenza virus surface glycoprotein neuraminidase NA.

[0035] Specific steps are as follows:

[0036] G418 selection of stable expression cell lines:

[0037] Before screening, determine the optimal concentration of G418 for screening MDCK cells is 500 μg / mL.

[0038] Preparation of G418: Dissolve 1g of G418 in 1mL of 1M HEPES solution, add ultrapure water to 10mL, filter, and store at 4°C for later use.

[0039] (1) The RNA fragment of the surface glycoprotein neuraminidase NA of type A influenza virus subtype H9N2 was reverse-transcribed in vitro to make cDNA, and the NA gene fragment of influenza virus was amplified with cDNA as a template, and cloned into the vector plasmid pD2EGFP- on N1. The plasmid pD2EGFP-NA was obtained.

[0040] (2) Spread MDCK cells on a 6-well plate and culture them in M...

Embodiment 3

[0052] Embodiment 3 tests the growth curve of recombinant virus

[0053] MDCK cells stably expressing NA were cultured in MEM medium containing 10% fetal bovine serum (Fetal Bovine Serum, FBS) and 1% double antibody (Penicillin-Streptomycin Solution, PS) in a 37°C incubator. (Media and serum were purchased from Biological Industries Company.) The transformed MDCK cells were plated in 6-well cell culture plates, 3×10 per well 5 cells, virus amplification was carried out when the cell growth density reached 90%. Before virus amplification, wash the transformed MDCK cells with phosphate buffer saline (Phosphate Buffer Saline, PBS) twice, infect the cells with the recombinant virus with MoI=0.001, and replace it with 2 mL containing 0.2% bovine serum albumin after 1 hour of adsorption. Protein (Bovine Serum Actin, BSA) and 1 μg / mL TPCK in MEM culture solution, and then the supernatant was collected at 12, 24, 48, and 72 hours after infection and stored at -80°C.

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Abstract

The invention provides an H5 subtype HA expressing replication-defective recombinant H9N2 avian influenza virus. The H5 subtype HA expressing replication-defective recombinant H9N2 avian influenza virus can provide protection against H5N1 subtype avian influenza and H9N2 subtype influenza simultaneously when serving as a vaccine and meanwhile enhance the safety of the attenuated vaccine by means of the characteristic of replication defection. The principle of the H5 subtype HA expressing replication-defective recombinant H9N2 avian influenza virus comprises that, under the premise of not changing hereditary stability, the structured recombinant avian influenza virus can simultaneously stably express the hemgglutinins (HA) of the H9N2 subtype avian influenza virus and the H5N1 subtype avianinfluenza virus, and only in MDCK (Madin-Darby canine kidney cells) capable of stably expressing the NA (neuraminidase) of the H9N2 subtype avian influenza virus, the recombinant H9N2 avian influenzavirus can replicate and successfully package recombinant virions; due to lack of structural protein NA genes, the recombinant avian influenza virus cannot achieve replication in human body, thereby avoiding body pathogenicity but inducing human body to generate high-level mucosal immune response and cellular immune response.

Description

technical field [0001] The invention belongs to the field of research and development of influenza virus vaccine technology, in particular to a replication-deficient recombinant H9N2 avian influenza virus expressing H5 subtype HA; Recombinant influenza virus of lectin HA, its preparation method and application. Background technique [0002] Avian influenza is a kind of infectious disease caused by type A influenza virus (Avian influenza virus, AIV) of poultry respiratory system disease and systemic sepsis. The H5N1 subtype avian influenza virus is a highly pathogenic virus that is highly contagious and seriously endangers poultry, humans and other animals. The H9N2 subtype is a low-pathogenic avian influenza virus, which exists widely in the world, and the damage is persistent and difficult to control, especially the mixed infection leads to high mortality. At the same time, the H9N2 subtype influenza virus is also the internal gene donor of H5N1, H7N9 and other subtype in...

Claims

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Application Information

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IPC IPC(8): C12N7/01A61K39/295A61K39/145A61P31/16
CPCA61K39/12A61P31/16C12N7/00A61K2039/5254A61K2039/70C12N2760/16121C12N2760/16134C12N2760/16151C12N2760/16122A61K39/145C12N7/04C12N2760/16034C12N2760/16061
Inventor 李军伟孙明宏吴叔文
Owner QINGDAO AGRI UNIV
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