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Fowl pox virus double gene expression vector (pg7.5n)

An expression vector, fowl pox virus technology, applied in the direction of gene therapy, genetic engineering, plant gene improvement, etc., can solve the problems of cumbersome operation, reduced expression of the second gene, and not many enzyme cutting sites, etc., to increase the number of , the effect of reducing the length of the vector and improving the expression efficiency

Inactive Publication Date: 2012-02-01
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] At present, the existing fowlpox virus vectors are not perfect, such as the vector is too large, the length of the foreign gene that can be carried is limited, there are not many available restriction sites, and the operation is cumbersome
Moreover, most of the double-gene expression vectors are expressed in cis, which has a certain reduction in the expression of the second gene.

Method used

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  • Fowl pox virus double gene expression vector (pg7.5n)
  • Fowl pox virus double gene expression vector (pg7.5n)
  • Fowl pox virus double gene expression vector (pg7.5n)

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Embodiment 1

[0031] This embodiment will take the expression vector shown in SEQ ID NO.1 as an example to describe its construction method and application in detail.

[0032] 1. Materials

[0033] Live fowlpox vaccine (I) was purchased from Qianyuanhao Nanjing Biopharmaceutical Factory. pGEM4z was purchased from Huamei Bioengineering Co., Ltd. 6-well cell culture plate, DMEM, Opti-MEM and Lipofatamine2000 were purchased from Invitrogen. X-gal, low melting point agarose, T4 polynucleotide kinase, PrimeSTAR TM HS DNA polymerase and DNA LigationKit (Mighty Mix) were purchased from Takara. Gel Extraction Kit and Plasmid miniKit (200) were purchased from Omega. JM109 competent cells were purchased from Takara. The primers used were the same as in the specification.

[0034] 2. Construction method

[0035] 2.1 Shuttle plasmid construction method

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Abstract

The present invention provides a fowlpox virus double-gene expression vector, which has the nucleotide sequence shown in SEQ ID NO.1, including promoter P7.5, LacZ gene, multiple cloning site on the LacZ gene, and rare enzyme Cutting site Not1, fowlpox virus DNA homology arms F11L1 and F11L2, AmpR resistance marker gene, origin of replication (ori). The dual-gene expression vector constructed by the present invention minimizes the length of the vector so that larger foreign genes can be accommodated; the number of available restriction sites is increased to facilitate the cloning of different foreign genes; the insertion site Not1 The promoter is not limited, and the promoter that matches with the exogenous gene can be freely selected, thereby improving the expression efficiency, and the expression efficiency of the exogenous gene can be improved by adopting the method of trans expression.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a fowlpox virus double-gene expression vector, its construction method and application. Background technique [0002] Poxviridae is a large group of obligate viruses with a diameter of 300-400nm and a lipid-protein shell surrounding a complex core structure. This structure contains a linear nearly 20kb double-stranded DNA molecule, encoding multiple subunits, and DNA-dependent The RNA polymerase, transcription factor, cap structure and methylase, poly A polymerase, are all packaged in the viral core and translated into mRNAs. Fowlpox Virus (FPV) belongs to the genus Fowlpoxvirus of the family Poxviridae. The virion core contains double-strand linear DNA, G+C=35%, and the genome is about 300kb long. Under natural conditions, it can only infect poultry, but it can produce abortive infection on mammalian cells, does not produce infectious virus particles, but can express a...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/863A61K48/00A61K31/7088
Inventor 廖明江经伟孔令辰郁宏伟任涛
Owner SOUTH CHINA AGRI UNIV
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