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Henpox virus universal transfer carrier, recombination henpox virus and its preparation method

A fowlpox virus and transfer vector technology, applied in the field of fowlpox virus universal transfer vector, can solve the problems of difficult application of recombinant virus, low expression efficiency, unfavorable recombinant virus screening, etc. expressive effect

Inactive Publication Date: 2007-05-02
POULTRY INST SHANDONG ACADEMY OF AGRI SCI
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Problems solved by technology

The promoter of the commercialized green fluorescent protein gene expression cassette is the CMV promoter, and the expression efficiency in the recombinant fowlpox virus is not too high, which is not conducive to the screening of recombinant viruses
More importantly, in the process of genetic manipulation and screening and purification of recombinants, screening markers such as luciferase and LacZ are often required, so the constructed recombinant virus genome contains these reporter genes, which brings great benefits to the application of recombinant viruses. must be difficult

Method used

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  • Henpox virus universal transfer carrier, recombination henpox virus and its preparation method
  • Henpox virus universal transfer carrier, recombination henpox virus and its preparation method
  • Henpox virus universal transfer carrier, recombination henpox virus and its preparation method

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Embodiment Construction

[0022] 1. Amplification and cloning of pPCI recombinant arm

[0023] Primer design: the positive strand primer is 5′-TAGAAACTCATACTCGTTACTG-3′, and the negative strand primer is 5′-CGATGAAGATATGTCTATACAC-3′. A fragment with a span of 2.35 kb was amplified, located between 34180 and 36534 of the fowlpox virus genome (GENBANK accession number AJ581527).

[0024] DNA extraction: inoculate monolayer CEF with fowlpox virus 282E4 strain (commercial vaccine), harvest cells after 96h, add equal volume lysate (containing 0.2mg / mL proteinase K, 1% SDS, 10mmol / LNaCl, 10mmol / LTris-HCl pH8.0, 1mmol / LEDTA), put in a water bath at 37°C for 3-4h. Extract the lysate with phenol, phenol:chloroform, and chloroform once each, add 2.5 times the volume of absolute ethanol, 1 / 10 volume of NaAc (5mol / LpH5.2), place at -20°C for 30min, centrifuge at 10000g for 10min to precipitate DNA, 70 Wash once with % ethanol, dissolve the precipitate in an appropriate amount of TE (pH 8.0), and store at -20°C f...

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Abstract

This invention relates to a universal transfer vector of poxvirus avium, recombinant poxvirus avium and its preparation. Duplicate nonessential region fragment of Poxvirus avium is used to amplificate and clone for pPCI carrier with PCR. Gene cassette of green fluorescent protein(GFP) is constructed with early or late composed promotor of avian molluscum roup virus, and loxP sequence is transferred into its two sides, and then inserting into pPCI together to obtain universal transfer plasmid vector pHBP, which together with Poxvirus avium genome DNA cotransfecting CEF to obtain recombinant virus rFPVGFP.

Description

technical field [0001] The universal fowlpox virus transfer carrier of the invention belongs to the high-tech field of biotechnology, and is specially used for the construction of exogenous gene recombinant fowlpox virus. Background technique [0002] Fowlpox virus is a member of the genus Fowlpoxvirus of the Poxviridae Vertebratepoxvirinae subfamily. Its host range is narrow and it only infects poultry. It presents abortive infection in mammals, but it does not affect the expression of foreign genes and DNA replication. . Therefore, it can not only be used to develop genetically engineered live vector vaccines for poultry, but also can be used as a non-replicating virus vector to develop mammalian genetically engineered live vector drugs for the prevention or treatment of diseases in animals other than poultry and even humans. The protective antigen genes of avian influenza virus, Newcastle disease virus and Marek's disease virus have been successfully expressed in fowl po...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/63C12N15/863C12N15/65
Inventor 王莉莉黄兵宋敏训于可响黄宝华
Owner POULTRY INST SHANDONG ACADEMY OF AGRI SCI
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