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A kind of avian influenza virus-like particle antigen, vaccine and its preparation method and application

An avian influenza virus, particle antigen technology, applied in the direction of virus antigen components, botanical equipment and methods, biochemical equipment and methods, etc.

Active Publication Date: 2022-06-14
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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  • A kind of avian influenza virus-like particle antigen, vaccine and its preparation method and application
  • A kind of avian influenza virus-like particle antigen, vaccine and its preparation method and application
  • A kind of avian influenza virus-like particle antigen, vaccine and its preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Embodiment 1: Construction of recombinant shuttle plasmid

[0048] 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). Primers are designed according to the nucleic acid sequences of the HA, NA, and M1 genes of the H7N9 GD15 strain of the avian influenza virus, and are used for gene amplification. The primers used in the construction were designed with Primer Premier 5.0 software and synthesized by Shanghai Bioengineering Technology Co., Ltd. The primer information is shown in Table 1.

[0049] Table 1 Primer information for amplifying the target...

Embodiment 2

[0055] Example 2: Rescue of recombinant baculovirus

[0056] The recombinant shuttle plasmid pVL-NA-HA-M1-GD15 and the optimized baculovirus genomic DNA were co-transfected into Sf9 cells in logarithmic growth phase. The brief procedure is as follows:

[0057] 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;

[0058] 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; add 5 μL to anot...

Embodiment 3

[0063] Example 3: Identification of recombinant baculovirus

[0064] 1 PCR identification of exogenous genes in recombinant baculovirus genome

[0065] 1.1 Extraction of recombinant viral DNA

[0066] The extraction of recombinant baculovirus DNA was carried out according to the instructions of the whole gold virus extraction kit:

[0067] 1) Mix Poly A Carrier RNA with Binding Buffer 1:50, called MIX A;

[0068] 2) For each sample, take 200 μL supernatant, add 200 μL BB5 and 20 μL proteinase K, and vortex to mix;

[0069] 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;

[0070] 4) Install the adsorption column, transfer the mixed solution to the column, centrifuge at 12000rpm for 1min, and discard the lower liquid;

[0071] 5) Add 500 μL WB5, centrifuge at 12000 rpm for 1 min, discard the filtrate, and repeat this step once;

[0072] 6) Centrifuge at 12000rpm for 1min, dis...

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Abstract

The invention discloses an avian influenza virus-like particle antigen, a vaccine and its preparation method and application. It is formed by self-assembly of a recombinant baculovirus transfected insect cells based on serial expression of H7N9 subtype avian influenza virus HA, NA, and M1 genes Bird flu virus-like particle antigen; the virus-like particle antigen prepared by the present invention is used to prepare vaccine. The vaccine prepared by the invention can produce higher titers of HI antibody, neutralizing antibody and IgY antibody after immunizing chickens, can provide 100% clinical protection against H7N9 subtype highly pathogenic avian influenza virus, and can effectively inhibit shedding . The invention does not rely on chicken embryos, has low production cost and short cycle; only one immunization with a low dose can induce strong protective ability. The invention provides a basis for the industrialized production of VLP vaccines.

Description

technical field [0001] The invention belongs to the technical field of vaccines, and in particular relates to an avian influenza virus-like particle antigen, a vaccine 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 virus...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/11C07K19/00C12N15/62C12N15/866A61K39/145A61P31/16
CPCC07K14/005C12N15/86A61K39/12A61P31/16C07K2319/00C12N2760/16122C12N2760/16123C12N2760/16134C12N2710/14043A61K2039/70A61K2039/5258
Inventor 胡娇刘秀梵李军李如梦胡增垒王晓泉顾敏
Owner YANGZHOU UNIV
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