Bird flu, Marek's disease dual live vaccine rMDV-HA viral strain and construction method thereof

A technology of Marek's disease and avian influenza virus, applied in the field of virus strains, can solve the problems of virus pollution, short immunity period, high production cost of inactivated vaccines, etc., and achieve the effect of stable virus and low production cost

Inactive Publication Date: 2009-11-11
YANGZHOU UNIV
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Problems solved by technology

Although inactivated vaccines can stimulate the body to produce humoral immunity, they cannot produce mucosal immunity and cellular immunity, so the immunit

Method used

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Embodiment Construction

[0013] The general idea of ​​the present invention is: using the MDV Rispens CVI 988 vaccine strain as a carrier to efficiently express the H5 subtype AIV HA, and construct a dual live vaccine strain of avian influenza and Marek's disease.

[0014] The invention includes: inserting REV LTR and HA gene of H5 subtype AIV SY strain into MDV Rispens CVI 988 vaccine strain genome to obtain purified rMDV-HA virus strain; detecting the situation of rMDV-HA virus strain expressing AIV HA.

[0015] The specific technical solutions adopted are as follows:

[0016] ①Amplification and cloning of DNA fragments

[0017] Using RT-PCR or PCR to amplify the HA gene of the H5 subtype AIV SY strain, the REV LTR, the screening marker gene lac / smGFP with loxP sites at both ends, and the foreign gene insertion site of the genome of the MDV Rispens CVI 988 vaccine strain Point the sequences on both sides, and clone these DNA fragments into plasmid vectors to obtain recombinant plasmids pHA, pLTR, p...

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Abstract

The invention relates to bird flu and Marek's disease dual live vaccine rMDV-HA viral strain and a construction method. H5 subtype bird flu virus SY strain haemagglutinin genes, reticuloendotheliosis disease virus LTR, screening mark genes lac/smGFP and Marek's virus Rispens CVI 988 vaccine strain genome exogenous genes are amplified by adopting reverse transcriptase-polymerase chain reaction or polymerase chain reaction and cloned to plasmid vectors to form recombinant plasmids pHA, pLTR, ploxP-lac/smGFP-loxP, pUP and pDOWN; DNA of transfer vector pMHA plasmids and DNA extracted from cells infected from the Marek's virus Rispens CVI 988 vaccine strain are co-transfected to chick embryo fibroblast; the exogenous genes are inserted to the Marek's virus Rispens CVI 988 vaccine strain genome through homologous recombination to form recombinant virus rMDV-HA/GFP with the screening mark genes; and the screening mark genes lac/smGFP of the rMDV-HA/GFP are removed through cre enzyme-mediated loxP site sequence recombination, and the rMDV-HA/GFP is purified to form the rMDV-HA viral strain. The rMDV-HA viral strain has the advantages of low cost, no pollution and long immunity period, and can be used as bird flu and Marek's disease dual live vaccine viral strain.

Description

technical field [0001] The invention relates to a virus strain, in particular to a rMDV-HA virus strain of a bird flu and Marek's disease dual live vaccine for poultry. Background technique [0002] Avian influenza and Marek's disease are two important infectious diseases of chickens, which are caused by avian influenza virus (AIV) and Marek's virus (MDV) respectively, which can cause great economic losses and public health hazards. [0003] Before the present invention, it has been found that the MDV Rispens CVI 988 vaccine strain has a good immune effect and a long immune period, and is currently a vaccine strain widely used in production. There are multiple non-essential segments for virus replication on the genome of the vaccine strain, which can be used as insertion sites for foreign genes when constructing recombinant viruses. [0004] The hemagglutinin (HA) on the AIV envelope is closely related to the pathogenicity of the virus. Sixteen HA subtypes of AIV have been...

Claims

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Application Information

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IPC IPC(8): C12N15/869C12N15/44A61K48/00
Inventor 吴艳涛郦晓琼张小荣徐晓静
Owner YANGZHOU UNIV
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