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Avian influenza virus-like particle antigen and preparation method and application thereof

An avian influenza virus, particle antigen technology, applied in the directions of virus antigen components, botanical equipment and methods, biochemical equipment and methods, etc. No biosafety risk, low production cost, good immunogenicity

Active Publication Date: 2019-09-17
LUOYANG HUIZHONG BIOTECH +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, because the mutation speed of avian influenza virus is much faster than the development and preparation speed of the corresponding vaccine of the mutant strain, and there is almost no cross-immune protection between different subtypes, the production of avian influenza vaccine needs to be constantly updated, and for influenza The production cycle of the vaccine corresponding to the epidemic strain of the virus is long and the production cost is high, resulting in a waste of manpower and material resources, and may not achieve the ideal prevention and control effect
[0005] Although the avian influenza virus-like particle vaccine published in literature research in the prior art can produce a better immune response, it still cannot reach the immune effect of commercial whole virus vaccines, and the general expression efficiency is low. Therefore, there is an urgent need for a A bird flu virus-like particle vaccine with good immune effect, safety and controllable cost

Method used

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  • Avian influenza virus-like particle antigen and preparation method and application thereof
  • Avian influenza virus-like particle antigen and preparation method and application thereof
  • Avian influenza virus-like particle antigen and preparation method and application thereof

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Embodiment approach

[0015] As an embodiment of the present invention, in the avian influenza virus-like particle antigen described in the present invention, the H5 subtype avian influenza virus HA, NA antigen protein gene and bovine immunodeficiency virus Gag antigen protein gene are respectively represented by the sequence SEQ.IDNo .1, represented by SEQ.ID No.3, SEQ.ID No.5 or represented by the sequence SEQ.ID No.2, SEQ.ID No.4, SEQ.ID No.6.

[0016] The present invention relates to an avian influenza virus-like particle vaccine, wherein the avian influenza virus-like particle vaccine comprises an immune amount of the avian influenza virus-like particle antigen and a pharmaceutically acceptable carrier.

[0017] The antigen of the avian influenza virus-like particle vaccine of the present invention is the self-assembly body of surface antigens hemagglutinin HA, neuraminidase NA and bovine immunodeficiency virus structural protein Gag of avian influenza virus. It can provide good immunogenicity...

Embodiment 1

[0045] The expression of embodiment 1 avian influenza HA protein

[0046] 1. Construction of Donor Plasmids

[0047] The HA gene shown in the sequence table SEQ.ID NO.1 was synthesized by Jinweizhi, and endonucleases BamHI and HindIII were added upstream and downstream of the gene, respectively. The synthetic HA gene was digested with BamHI and HindIII, ligated with the pFastBacI vector digested with the same restriction enzymes, and the ligated product was transformed into Escherichia coli DH5α, and the identified correct plasmid was named pFastBac-HA.

[0048] 2. Construction and identification of recombinant Bacmid

[0049] Add 2μl pFastBac-HA plasmid to DH10Bac competent cells, flick and mix well, incubate on ice for 30min, heat shock at 42℃ for 45s, incubate on ice for 5min, add 400μl of SOC medium at 37℃200rpm for 4h, take 100μl of bacterial liquid to spread Incubate at 37°C for at least 48 hours on a plate containing IPTG / X-gal / Kana / Tetracycline / Golden Third Antibody,...

Embodiment 2

[0055] Example 2 High-efficiency expression of avian influenza HA protein

[0056] The high-efficiency expression sequence was optimized according to the HA gene shown in the sequence table SEQ.ID NO.1, and the optimized sequence was shown in the sequence table SEQ.ID NO.2.

[0057] The high-efficiency expression of avian influenza HA protein was carried out with reference to the method of Example 1. It was determined that the HA content in the supernatant was 0, and the HA content in the cells was 12log2. Observation by transmission electron microscopy showed that protein fragments aggregated and no virus-like particles were formed.

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Abstract

The invention relates to an avian influenza virus-like particle antigen. The avian influenza virus-like particle antigen consists of H5 subtype avian influenza virus HA and NA antigens of and a bovine immunodeficiency virus Gag antigen. The invention also relates to a preparation method of the particle antigen and a vaccine prepared by using the particle antigen. The avian influenza virus-like particle antigen can be effectively assembled when prepared by using insect cells, and appears as virus-like particles. The vaccine prepared by using the avian influenza virus-like particle antigen can effectively provide effective protection against H5 subtype and / or H9 subtype avian influenza virus, and the protection effect of the vaccine is equivalent to or better than that of inactivated vaccines with the same HA antigen content.

Description

technical field [0001] The invention relates to the field of biopharmaceuticals, in particular to a virus-like particle antigen for protection of avian influenza virus, its preparation method and application. Background technique [0002] Virus-like particles (VLPs) are hollow particles between 15nm and 400nm assembled by structural proteins of viruses. VLPs can be prepared by highly expressing one (or several) structural proteins of a certain virus in vitro, so that they can automatically assemble into hollow particles similar in shape to natural viruses. The method is mainly to clone viral structural protein genes into expression vectors, and then transfer these vectors into prokaryotic or eukaryotic cells for expression. [0003] Avian Influenza Virus (AIV) belongs to the family Orthomyxoviridae, the genus Influenzavirus, and is a type A influenza virus. Avian influenza (Avian Influenza, AI) is a bird infection and disease syndrome caused by the virus. The Internationa...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/145A61P31/16C12N7/00C12N15/866C12R1/93
CPCA61K39/12C12N7/00C12N15/86A61K2039/5258C12N2760/16134C12N2760/16123C12N2710/14043Y02A50/30
Inventor 田克恭张盼涛王同燕孙进忠张许科
Owner LUOYANG HUIZHONG BIOTECH
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