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

An avian influenza virus and particle technology, applied in the fields of botanical equipment and methods, biochemical equipment and methods, viruses, etc., can solve problems such as failure to achieve prevention and control effects, high production costs, and long production cycles, and achieve no biological safety. Risk, low production cost, good immunogenicity

Active Publication Date: 2021-10-01
PU LIKE BIO ENG +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, since 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 the vaccine against influenza virus The production cycle of the vaccine corresponding to the epidemic strain 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 the 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, screening ideal It is urgent to prepare an avian influenza virus-like particle vaccine with good immune effect, safety and controllable cost. It also meets the needs of the country to effectively prevent and control major animal diseases and ensure the healthy and sustainable development of animal husbandry.

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 1

[0045] Example 1 Expression of H5 subtype avian influenza virus-like particles

[0046] 1. Carrier modification

[0047] Using the commercialized vector pFastBac I as a template, NdeI, NotI, SalI and XbaI restriction enzyme sites were sequentially inserted at positions 4413-4414. Add 1 μl of DpnI enzyme to the PCR product to digest the template plasmid, take 5 μl and transform it into DH 5α according to the conventional method, and the successfully transformed plasmid is named pFastBac mut.

[0048] 2. Construction of three expression cassette donor plasmids

[0049] The HA gene shown in Seq ID NO.1, the NA gene shown in Seq ID NO.2, and the M1 gene shown in Seq ID NO.3 were synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd., and the upstream and downstream of the genes were respectively added Dicer BamHI and HindIII restriction sites. The synthesized HA gene, NA gene, and M1 gene were respectively digested with BamHI and HindIII, and then ligated with the pFastBac mut...

Embodiment 2

[0059] Example 2 Preparation of H5 subtype avian influenza virus-like particle vaccine

[0060] The virus-like particles harvested in Example 1 were respectively added to the mineral oil adjuvant to prepare various vaccine compositions, and the specific proportions are shown in Table 1.

[0061] Table 1 Proportion of H5 subtype avian influenza virus-like particle vaccine composition

[0062] components Vaccine 1 Vaccine 2 Vaccine 3 Vaccine 4 Vaccine 5 Vaccine 6 rBac-M1-1 (HA content) 6log2 8log2 - - 6log2 8log2 rBac-M1-2 (HA content) - - 6log2 8log2 6log2 8log2 Adjuvant (V / V%) 66％ 66％ 66％ 66％ 66％ 66％

Embodiment 3

[0063] Example 3 H5 subtype avian influenza virus-like particle vaccine immunogenicity test

[0064] Get 80 SPF chickens at the age of 21 days, divide them into 8 groups, 10 in each group, the 1st group to the 6th group are respectively injected vaccine 1~vaccine 6 prepared in Example 2 through the neck subcutaneously, and the 7th group subcutaneously injects commercial products Chemical inactivated vaccine (H5 (Re-11+Re-12), H7 (H7-Re2) trivalent inactivated vaccine, H5HA content is 8log2), immunization dose is 0.3ml, the 8th group is subcutaneously injected with 0.3ml normal saline , as a blank control. All experimental chickens were kept in isolation, blood was collected on the 14th and 21st day after immunization, serum was separated, and the titer of HI antibody was determined*. The test results of different HI antibodies after immunization are shown in Tables 2 and 3.

[0065] Table 2 H5 subtype avian influenza virus-like particle vaccine immunogenicity test results 1 ...

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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 H5 subtype and / or H7 subtype avian influenza virus-like particle antigen and a pharmaceutically acceptable carrier, and the avian influenza virus-like particle antigen is formed by assembling HA, NA and M1 antigen proteins of H5 subtype and / or H7 subtype avian influenza viruses. The avian influenza virus-like particle vaccine disclosed by the invention has good immunogenicity, and the immune effect is better than that of a commercial inactivated vaccine with higher antigen content when the dosage is low.

Description

technical field [0001] The invention relates to the field of biopharmaceuticals, in particular to a virus-like particle vaccine 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 virus. The I...

Claims

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Application Information

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IPC IPC(8): C07K14/11C12N15/866A61K39/145A61P31/16
CPCC07K14/005C12N15/86A61K39/12A61P31/16C12N2760/16123C12N2760/16122C12N2760/16134C12N2760/16152C12N2710/14043C12N2800/105A61K2039/5258A61K2039/552
Inventor 田克恭王同燕张盼涛张许科
Owner PU LIKE BIO ENG
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