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H3N8 subtype equine influenza recombinant virus-like particle vaccine as well as preparation method and application thereof

A recombinant virus, H3N8 technology, applied in the field of bioengineering, can solve problems to be tested in practice

Inactive Publication Date: 2014-07-02
HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The molecular epidemiological investigation of EIV in my country shows that EIV in my country and foreign imported vaccine strains belong to two different lineages in evolution: Florida clade 2 and Kentucky clade. Provide effective prevention and control of the epidemic? yet to be tested in practice

Method used

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  • H3N8 subtype equine influenza recombinant virus-like particle vaccine as well as preparation method and application thereof
  • H3N8 subtype equine influenza recombinant virus-like particle vaccine as well as preparation method and application thereof
  • H3N8 subtype equine influenza recombinant virus-like particle vaccine as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1 Construction of recombinant baculovirus rBV-XJ3-HA+M1 expressing EIV HA and M1 protein of the present invention

[0034] The preparation flow chart of expressing EIV HA and M1 recombinant baculovirus rBv-XJ3-HA+M1 is as follows figure 1 shown.

[0035] 1. RT-PCR amplification of EIV XJ3HA and M1 genes

[0036] EIV XJ3 was extracted using the Huashun column virus RNA extraction kit (this strain has been recorded in the literature: H3N8 subtype EIV HA gene expression and biological characteristics analysis in insect cells, Liu Chunguo et al., "Chinese Journal of Preventive Veterinary Medicine" 2013 In the 11th issue of the year, it is now preserved and provided by the Harbin Veterinary Research Institute of the Chinese Academy of Agricultural Sciences. ), the specific steps refer to the kit instructions. XJ3 cDNA was prepared by using the universal primer Uni-12: 5'-AGCAAAAGCAGG-3' for reverse transcription of influenza A virus as the reverse transcription pri...

Embodiment 2

[0056] Example 2 Identification of recombinant baculovirus rBV-XJ3-HA+M1 expressing EIV HA and M1 proteins of the present invention

[0057] 1. Detection of protein expression by cell immunohistochemistry:

[0058] The 4th generation rBV-XJ3-HA+M1 and wtBV obtained in Example 1 were respectively inoculated into Sf9 cells cultured in 6-well plates for immunohistochemical test (Liu Chunguo et al., Progress in Natural Science, 2009, 19 (8): 812-818.). The main steps are as follows: 3 days after virus inoculation, the supernatant was discarded, the cells were washed 3 times with pre-cooled PBS, and then fixed with 4% formalin for 10 min. Mouse anti-H3N8 subtype EIV-specific polyclonal serum (1:200) was used as the primary antibody, and goat anti-mouse IgG-HRP (1:2000) was used as the secondary antibody. After DAB color development, rBV- Sf9 cells infected with XJ3-HA+M1 showed positive brown staining, while Sf9 cells inoculated with wtBV showed no staining, the results were as f...

Embodiment 3

[0065] Example 3 Preparation of the recombinant virus-like particle vaccine of the present invention and its immune protection experiment on mice

[0066] 1. Purification of virus-like particles

[0067] The fourth-generation recombinant baculovirus rBV-XJ3-HA+M1 obtained in Example 1 was inoculated into Sf9 cells, cultured at 27°C for 72 hours, and harvested after repeated freezing and thawing for 3 times, centrifuged at 3,000 r / min for 10 minutes to remove cell debris. Supercentrifuge the supernatant at 38,000r / min (100,000×g) for 2h, then use 20%, 30% and 60% sucrose pads for density gradient centrifugation, collect the protein between the 30% and 60% sucrose pads and desugar , and the precipitate was resuspended with TNE to obtain purified virus-like particles.

[0068] 2. Quantification of virus-like particles

[0069] The concentration of total protein was determined by the Bradford method, and the specific method is shown in the instruction manual. Then use SDS-PAGE ...

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Abstract

The invention discloses an H3N8 subtype equine influenza recombinant virus-like particle vaccine as well as a preparation method and an application thereof. The vaccine in the invention contains recombinant virus-like particle protein which is obtained by expressing nucleotide sequences as shown in SEQ ID NO.1 and SEQ ID NO.2 through an insect / baculovirus expression system, a pharmaceutically acceptable carrier, an adjuvant or an excipient. The recombinant virus-like particle has a morphological structure similar with that of a natural virus particle, and has good immunogenicity; the vaccine prepared by the recombinant virus-like particle can induce a mouse to generate good protective immune response after immunizing the mouse, and is free of any risk of live virus; and meanwhile, naturally infected persons and immune persons can be distinguished, so that epidemiological investigation is not affected, and therefore, the H3N8 subtype equine influenza recombinant virus-like particle vaccine disclosed by the invention can be used as a candidate vaccine for preventing and treating the equine influenza.

Description

technical field [0001] The invention relates to an equine influenza vaccine and its preparation method and application, in particular to a H3N8 subtype equine influenza recombinant virus-like particle vaccine and its preparation method and application. The invention belongs to the technical field of bioengineering. Background technique [0002] Equine influenza (Equine influenza, EI) is a severe acute high-level contact infection of the upper respiratory tract of equine animals caused by equine influenza virus (Equine influenza virus, EIV) in the Orthomyxoviridae, Influenzavirus type A influenza virus Diseases are often explosive and spread rapidly and widely, causing serious economic losses to the horse breeding and horse racing industries. OIE stipulates that equine influenza is a legally notifiable animal disease, and my country lists it as a third-class infectious disease. [0003] In recent years, EI has frequently broken out all over the world, and my country is no ex...

Claims

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Application Information

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IPC IPC(8): C12N7/04C12N15/866A61K39/145A61P31/16
Inventor 刘明刘春国相文华
Owner HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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