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Plasmid-type adenovirus vector pAd-PPARGC1A and construction method thereof

An adenovirus and vector technology, applied in the field of genetic engineering, can solve the problems of difficult operation, no research report, and the genome is too large, and achieve the effect of ensuring biological safety.

Inactive Publication Date: 2012-11-07
NORTHWEST A & F UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] 2) The genome is too large (36kb) and difficult to manipulate
[0010] There is no research report on the function of PPARGC1A gene in cattle

Method used

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  • Plasmid-type adenovirus vector pAd-PPARGC1A and construction method thereof
  • Plasmid-type adenovirus vector pAd-PPARGC1A and construction method thereof
  • Plasmid-type adenovirus vector pAd-PPARGC1A and construction method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1 , Construction of pAd-PPARGC1A plasmid:

[0043] 1. Materials and methods

[0044] 1.1. Instrument:

[0045] Ultra-clean bench, biochemical incubator, gene amplification instrument, PTC-200 single-slot gradient gene amplification instrument, Heraeus refrigerated high-speed centrifuge (Germany), Bio-Rad gel imaging analyzer (USA), CO 2 Incubator, fluorescence microscope, HZS-H water bath shaker (Harbin), Eppendorf pipette, DYY-III steady voltage and steady flow electrophoresis instrument (Beijing Liuyi), DYY-III31A and DYY-III28D electrophoresis tank (Beijing Liuyi ), ice maker, MDF-382E ultra-low temperature refrigerator (Sanyo, Japan), Eppendorf desktop high-speed centrifuge, Sartorious electronic balance (Germany), conventional refrigerator, etc.

[0046] 1.2. Main biochemical reagents and kits:

[0047] PrimeSTAR DNA polymerase, DNA restriction enzymes (Sal I, Not I, Pac I, Pme I, etc.), trypsin, DNA Marker, T4 DNA ligase, Trizol, reverse transcription...

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Abstract

The invention relates to a plasmid-type adenovirus vector pAd-PPARGC1A (peroxisome proliferator-activated receptor gamma coactivator 1 alpha) and a construction method thereof, and belongs to the technical field of gene engineering. The construction method is as below: inserting a Qinchuan cattle PPARGC1A gene under a CMV promoter of a pAdTrack-CMV shuttle plasmid to construct a recombinant shuttle plasmid pAdTrack-CMV-PPARGC1A; linearizing by PmeI and transforming E.coli BJ5183 competent bacteria containing pAdEasy-l adenovirus backbone plasmids; carrying out homologous recombination on the pAdTrack-CMV-PPARGC1A and the pAdEasy-1 in bacteria, by using an efficient homologous recombination system of an E.coli BJ5183 endogenous Cre recombinase; naming a correct recombinant adenovirus plasmid after pAd-PPARGC1A; linearizing the pAd-PPARGC1A by Pac I and exposing inverted terminal repeats (ITR); and transfecting HEK-293A cells by the liposome to produce recombinant virus particles. The present invention has advantages of ensuring biological safety of the recombinant adenovirus vector, promoting efficient expression of the gene and benefiting expression of the gene for subsequent detection.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering and relates to a plasmid-type adenovirus vector pAd-PPARGC1A and a construction method thereof, in particular to a plasmid-type adenovirus vector containing a cattle PPARGC1A gene and a construction method of the plasmid-type adenovirus vector. Background technique [0002] The AdEasyTM system is a shortcut system constructed by T.C.He et al. (1998) to replace the traditional adenovirus recombination system. In this system, recombinant adenoviruses can be produced in only two steps: first, the expression cassette is loaded into the shuttle vector, and then inserted into the adenoviral genome by homologous recombination. The most effective way to insert foreign genes into adenovirus is through homologous recombination, because: 1) adenovirus DNA is a large linear molecule containing almost all restriction enzyme sites; [0003] 2) The genome is too large (36kb) and difficult to manipul...

Claims

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Application Information

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IPC IPC(8): C12N15/861C12N15/66
Inventor 陈宏李密杰刘栋蓝贤勇雷初朝
Owner NORTHWEST A & F UNIV
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