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| Construction and Identification of Recombinant Adenovirus Vector Specific to Qinchuan Cattle(Bos taurus) Derived Sterol Regulatory Element Binding Protein 2 Gene (SREBP2) |
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Abstract Sterol regulatory element binding protein 2(SERBP2) is a basic-helix-loop-helix-luecine zipper factor which regulates the metabolization process of cholesterol. We cloned the SREBP2 gene from Qinchuan cattle (Bos taurus) and constructed the overexpression adenoviral vector, and packed and amplified the virus for a high titer, as a antecedent work for the further study of cellular level function of SERBP2 gene. Total RNA was extracted from the adipose tissue of Qinchuan cattle and then reversely transcripted to cDNA. A pair of exclusive primers were designed according to the GenBank sequence information of SREBP2 gene(Accession No. NM_001205600) to amplify the complete coding sequence(CDS) area of SREBP2 gene by polymerase chain reaction (PCR). The fragments containing CDS area of SREBP2 gene were inserted into the shuttle vector to construct the pAdTrack-CMV-SREBP2 plasmid. The recombinant plasmid and the blank control pAdTrack-CMV were linearized by digesting with restriction endonuclease PmeⅠ and subsequently transformed into Escherichia coli BJ5183 containing pAdEasy-1 to homologous recombine and obtain the recombinant adenovirus plasmid pAd-SREBP2 and pAd-CMV. And then, the confirmed recombinant adenovirus plasmid pAd-SREBP2 was digested with PacⅠand transfected into 293A cell line to package and amplify the recombinant adenovirus Ad-SREBP2 and Ad-CMV, and to collect virus of high titer. The viral titer of Ad-SREBP2 and Ad-CMV was 7×108 and 1.3×109 GFU/mL respectively, measured by green fluorescent protein (GFP) labelled method. Qinchuan cattle derived preadipocyte was infected by Ad-SREBP2 and Ad-CMV to verify the availability of the virues. The expression of SREBP2 increased by 102.3 times after infected with the recombinant adenovirus for 48 h, determined by quantitative Real-time PCR. The cloning of SERBP2 gene of Qinchuan cattle obtaining of recombinant adenoviru and virus of high titer are set as foundation for the studies of the gene function on cellular level.
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Received: 03 April 2013
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