Recombinant H7N9 subtype avian influenza virus-like particle, and preparation method and application thereof

A bird flu virus and particle technology, applied in the field of vaccines, can solve problems such as insufficient supply of chicken embryos, endogenous virus contamination, environmental pollution, etc.

Active Publication Date: 2020-12-15
YANGZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, traditional inactivated vaccines rely on chicken embryos for production, which has disadvantages such as insufficient supply of chicken embryos during disease outbreaks, and the production of a large amount of waste causing environmental pollution and endogenous virus contamination.
In addition, inactivated vaccines can only induce antibody immunity but not cellular immunity

Method used

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  • Recombinant H7N9 subtype avian influenza virus-like particle, and preparation method and application thereof
  • Recombinant H7N9 subtype avian influenza virus-like particle, and preparation method and application thereof
  • Recombinant H7N9 subtype avian influenza virus-like particle, and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Embodiment 1: Construction of recombinant shuttle plasmid

[0054] The H7N9 subtype avian influenza virus A / Chicken / Guangdong / GD15 / 2016 (hereinafter referred to as GD15 strain) used in this experiment was preserved and provided by the Key Open Laboratory of Livestock and Poultry Infectious Diseases, Ministry of Agriculture, Yangzhou University. The strain has been published on the genebank sequence, the sequence number is: PB2 (KY751288), PB1 (KY751256), PA (KY751233), HA (KY751058), NP (KY751157), NA (KY751124), M (KY751091), NS (KY751190). According to the nucleic acid sequences of the HA, NA, and M1 genes of the avian influenza virus GD15 strain, the Primer Premier 5.0 software was used to design primers for gene amplification.

[0055] Table 1 Primer information for amplification of HA, NA and M1 genes

[0056]

[0057] Note: GCCACC (bold) represents the Kozak sequence; the underlined line is the restriction site.

[0058] The PCR reaction system is as follows...

Embodiment 2

[0066] Example 2: Rescue of recombinant baculovirus

[0067] The recombinant shuttle plasmids pVL-HA-GD15, pVL-NA-GD15, pVL-M1-GD15 and optimized baculovirus genomic DNA were co-transfected into Sf9 cells in logarithmic growth phase. The brief procedure is as follows:

[0068] 1) Add Sf9 cells to the six-well cell plate, 1×10 6 Place the cell plate horizontally in a 27°C incubator, incubate for 1 hour, and prepare transfection samples at the same time;

[0069] 2) Prepare the DNA transfection complex: Dilute the recombinant shuttle plasmid to a concentration of 100ng / μL for use; warm the transfection reagent to room temperature and mix gently; prepare two sterile EP tubes, add 100 μL serum-free respectively Culture medium; add 5μL (100ng) flashBAC to one tube TM For DNA, flick the EP tube to mix evenly; add 5 μL (500ng) transfer vector plasmid, flick the tube to mix evenly, then add 4 μL of transfection reagent, flick the EP tube to mix evenly, and centrifuge to precipitate;...

Embodiment 3

[0074] Example 3: Identification of recombinant baculovirus

[0075]1 PCR identification of exogenous genes in recombinant baculovirus genome

[0076] 1.1 Extraction of recombinant viral DNA

[0077] The extraction of recombinant baculovirus DNA was carried out according to the instructions of the whole gold virus extraction kit:

[0078] 1) Mix Poly A Carrier RNA with Binding Buffer 1:50, called MIX A;

[0079] 2) For each sample, take 200 μL supernatant, add 200 μL BB5 and 20 μL proteinase K, and vortex to mix;

[0080] 3) Incubate at 56°C for 15 minutes, add 250 μL of absolute ethanol, vortex and oscillate to mix, and then place at room temperature for 15 minutes;

[0081] 4) Install the adsorption column, transfer the mixed solution to the column, centrifuge at 12000rpm for 1min, and discard the lower liquid;

[0082] 5) Add 500 μL WB5, centrifuge at 12000 rpm for 1 min, discard the filtrate, and repeat this step once;

[0083] 6) Centrifuge at 12000rpm for 1min, disc...

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Abstract

The invention discloses a recombinant H7N9 subtype avian influenza virus-like particle, and a preparation method and application thereof. Recombinant baculoviruses for expressing HA protein, NA protein and M1 protein of an H7N9 highly pathogenic avian influenza virus are respectively constructed on the basis of a recombinant baculovirus insect cell culture system. The three recombinant baculovirusstrains are used for co-infecting suspended insect cells, so that the H7N9 subtype avian influenza virus-like particle capable of being self-assembled in the cells can be obtained. After the concentration and purification with cane sugar with different gradient concentrations through an ultrafiltration tube, the H7N9 subtype avian influenza virus-like particle is mixed and emulsified with an adjuvant to prepare a vaccine. When a chicken is immunized by the prepared vaccine, the body can be induced to generate a specific antibody; and the advantages of high immunogenicity, good safety, high hereditary stability and the like are realized. After the attack by a lethal dose of H7N9 subtype highly pathogenic avian influenza viruses, the complete clinical protection can be provided, and virus expelling of the chicken is obviously inhibited. The invention provides a novel method for preventing H7N9 subtype avian influenza virus infection, and also lays a foundation for the development of a novel influenza virus vaccine.

Description

technical field [0001] The invention belongs to the technical field of vaccines, and in particular relates to a recombinant H7N9 subtype avian influenza virus-like particle and a preparation method and application thereof. Background technique [0002] Avian influenza virus (Avian Influenza Virus, AIV) belongs to type A influenza virus and is a single-stranded negative-sense RNA virus with 8 segments, which can cause disease and death of one or more birds and other animals. In 2013, the H7N9 subtype of avian influenza first appeared in the Yangtze River Delta region of my country. So far, five waves of epidemic peaks have been formed in the Chinese population, posing a major threat to public health and poultry farming. Found in the fifth wave of the epidemic, four basic amino acid residues (KRKRTAR / G or KGKRIAR / G) were inserted at the cleavage site connecting the HA1 and HA2 peptide regions, and the presence of multiple amino acids proves that H7N9 AIV has evolved from LPAI ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/11C12N15/62C12N15/866A61K39/145A61P31/16
CPCC07K14/005C12N15/86A61K39/12A61P31/16C12N2760/16122C12N2760/16123C12N2760/16134C12N2710/14043A61K2039/70A61K2039/5258
Inventor 胡娇刘秀梵李如梦李军胡增垒王晓泉顾敏
Owner YANGZHOU UNIV
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