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Avian influenza virus-like particle vaccine as well as preparation method and application thereof

A bird flu virus and particle technology, applied in biochemical equipment and methods, viruses, vaccines, etc., can solve the problems of not achieving the immune effect of commercial whole virus vaccines, not achieving the effect of prevention and control, and generally low expression efficiency, etc. Achieve good immune effect, excellent immune effect, and good persistence

Active Publication Date: 2019-12-13
LUOYANG HUIZHONG BIOTECH +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, because there is almost no cross-immune protection between different subtypes of avian influenza viruses, the current production of avian influenza vaccines cannot meet clinical needs, and the production cycle of corresponding vaccines for different subtypes of influenza viruses is relatively long and the production cost is relatively long. Higher, resulting in 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 vaccine as well as preparation method and application thereof
  • Avian influenza virus-like particle vaccine as well as preparation method and application thereof
  • Avian influenza virus-like particle vaccine as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1H7

[0056] Expression of embodiment 1H7 subtype avian influenza HA protein

[0057] 1. Construction of Donor Plasmids

[0058] For the HA gene shown in SEQ.ID NO.1 in the sequence table synthesized by Jinweizhi, endonucleases BamHI and HindIII restriction sites were added to the 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.

[0059] 2. Construction and identification of recombinant Bacmid

[0060] 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 / Tetra...

Embodiment 2H7

[0066] The expression of embodiment 2H7 subtype avian influenza virus-like particles

[0067] 1. Carrier modification

[0068] Using the commercially available vector pFastBac I as a template, use primers pFBmut-F and pFBmut-R to sequentially insert NdeI, NotI, SalI and XbaI restriction enzyme sites at positions 4413-4414 of pFastBacI. The primers are shown in Table 1. Add 1 μl of DpnI enzyme to the PCR product to digest the template plasmid, take 5 μl and transform it into DH5α according to the conventional method, and the successfully transformed plasmid is named pFastBac mut.

[0069] Table 1 Vector Transformation Primer List

[0070]

[0071] 2. Construction of three expression cassette donor plasmids

[0072] The HA gene shown in SEQ.ID NO.1, the NA gene shown in SEQ.ID NO.2, and the Gag gene shown in SEQ.ID NO.3 were synthesized by Jinweizhi in the sequence table. Enzyme BamHI and HindIII restriction sites. The synthesized HA gene, NA gene, and Gag gene were diges...

Embodiment 3H7

[0081]The preparation of embodiment 3H7 subtype avian influenza subunit vaccine and virus-like particle vaccine

[0082] The HA protein harvested in the cells of Example 1, the HA, NA, and Gag proteins harvested in the cells of Example 2, and the virus-like particles harvested from the extracellular supernatant of Example 2 with different contents were added to the white oil adjuvant to prepare See Table 2 for the specific proportions of each vaccine composition.

[0083] Table 2H7 subtype avian influenza vaccine composition ratio

[0084] components Vaccine 1 Vaccine 2 Vaccine 3 Vaccine 4 Embodiment 1 protein (HA content) 8log2 - - - Embodiment 2 protein (HA content) - 8log2 - - Embodiment 2VLPs (HA content) - - 6log2 8log2 White oil adjuvant (V / V%) 66％ 66％ 66％ 66％

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Abstract

The invention relates to an avian influenza virus-like particle vaccine. The avian influenza virus-like particle vaccine comprises an immune dose of H7 subtype avian influenza virus-like particle antigen and a pharmaceutically acceptable carrier, wherein the avian influenza virus-like particle antigen is formed by assembling H7 subtype avian influenza virus HA, NA antigen protein and bovine immunodeficiency virus Gag antigen protein. Compared with a subunit vaccine, the avian influenza virus-like particle vaccine has the advantages that the immune efficacy is greatly improved, and an inactivated vaccine with higher antigen content has a better immune effect. Meanwhile, protection can be provided for the H9 subtype avian influenza virus.

Description

technical field [0001] The invention relates to the field of biopharmaceuticals, in particular to a virus-like particle vaccine capable of providing protection against 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 vi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/145A61P31/16A61K39/21
CPCA61K39/12A61P31/16A61K2039/5258A61K2039/552C12N2740/15034C12N2760/16134Y02A50/30
Inventor 田克恭王同燕张盼涛孙进忠张许科
Owner LUOYANG HUIZHONG BIOTECH
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