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Fowl pos virus expression vector P12 18 and its construction process

A fowl pox virus and expression vector technology, applied in the field of fowl pox virus expression vector, can solve the problems of affecting immune efficacy and the like

Inactive Publication Date: 2007-11-07
YANGZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the destruction of ORF073 may be the main reason for affecting the immune efficacy of recombinant FPV with FPV7S as the insertion site of the foreign gene in commercial chickens with maternal antibodies

Method used

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  • Fowl pos virus expression vector P12 18 and its construction process
  • Fowl pos virus expression vector P12 18 and its construction process
  • Fowl pos virus expression vector P12 18 and its construction process

Examples

Experimental program
Comparison scheme
Effect test

Embodiment Construction

[0088] 1. Screening of the non-essential region for new replication of fowlpox virus FPV12-18 by directional cloning

[0089] Propagation of virus and extraction of genome

[0090] SPF chicken embryos of 9-10 days old were taken according to conventional methods to make CEF single layer. Inoculate 282E4 strain FPV in 5% CO 2 , 37 ° C incubator culture. Discard the original medium when the lesion reaches more than 80%, wash the cells twice with PBS, add an appropriate amount of PBS, and blow the cells down with a pipette. Centrifuge at low speed to discard the supernatant, and suspend with a small amount of PBS (200 μl for a 50 ml cell culture flask). The extraction method was carried out according to the instructions of the High Pure PCRTemplate Preparation Kit: Add 200 μl of the above sample, 200 μl of Binding Buffer and 40 μl of Proteinase K to a 1.5ml Eppendorf tube in sequence, and mix well; after 10 minutes at 72°C, add 100 μl of isopropanol; mix the above mixture Aft...

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Abstract

The present invention relates to fowl pos virus expression vector constructing technology, and is especially fowl pos virus expression vector p12-18 and its construction process. The fowl pos virus expression vector p12-18 constructing process includes the following steps: screening newly copied nonessential regions of fowl pos virus and amplifying fowl pos virus to copy two flanking regions of nonessential region FPV12-18; cloning these two flanking regions and marker gene expressing frame P11-LacZ successively into vector pBluescript SK(-), cotransfecting chick embryo fibroblast with the obtained vector pPl2 and FPV of 282E4 strain, and screening the recombinant virus with the marker gene of colibacillus beta-galactosidase LacZ gene to obtain recombinant virus vector; and finally inserting the exogenous gene promoter Ps into the vector to obtain fowl pos virus expression vector. The present invention can obtain recombinant fowl pos virus vaccine with high capacity.

Description

technical field [0001] The invention relates to a fowlpox virus expression vector which is beneficial to improve the ability of recombinant fowlpox virus to resist the interference of maternal antibody, especially the non-essential region for replication in the vector, so as to provide a kind of fowlpox virus capable of improving the ability of recombinant FPV vaccine to resist the interference of maternal antibody Methods. Background technique [0002] Fowlpox Virus (FPV) is an expression vector with unique advantages and broad application prospects following vaccinia virus VV, and is one of the research hotspots in the field of molecular biology today. Fowlpox virus belongs to the family Poxviridae, the genus Fowlpoxvirus, and Fowlpox virus is its representative species. FPV has its unique advantages as an expression vector. Its genome is huge, about 1.5 times the length of VV, and contains more non-essential regions for replication, which can be used to construct FPV mu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/863C12N15/66
Inventor 刘秀梵孙蕾陈素娟刘武杰
Owner YANGZHOU UNIV
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