秦川牛SREBP1基因重组腺病毒载体的构建与病毒包装

doi:10.11843/j.issn.0366-6964.2013.08.022

Abstract

Abstract:

The objective of this research was to construct recombinant adenovirus vector specific to SREBP1 gene of Qinchuan cattle and further package and amplify recombinant adenovirus carrying SREBP1 gene, aimed at studying SREBP1 gene function at cellular level. A pair of exclusive primers was designed according to the GenBank sequence information of SREBP1 gene to amplify the complete CDS area of SREBP1 gene via polymerase chain reaction (PCR). The obtained PCR products were then sub-cloned into pMD19-T simple vector. The confirmed fragments containing CDS area of SREBP1 gene were first digested from clone vector and then?insert into the shuttle vector to construct the pAd-Track-CMV-SREBP1 plasmid. The resultant plasmid was linearized by digesting with restriction endonuclease PmeⅠ and subsequently transformed into BJ5183 containing pAdEasy-1 to obtain the expression vector pAd-SREBP1. Overall, the confirmed recombinant adenovirus plasmid pAd-SREBP1 was digested with PacⅠ and transfected into 293A cell line to package and amplify the recombinant adenovirus. The viral titer was determined by GFP labeled method. SREBP1 gene was cloned from Qinchuan cattle with two mutations in the DNA fragments. The two mutations were proved to be caused probably by the varietal difference, not by external factors. Recombinant plasmid pAdTrack-CMV-SREBP1 and pAd-SREBP1 were successfully constructed to the package and amplify the recombinant adenovirus with 1.5×10⁹ GFU·mL^－1 as its titer.

CLC Number:

S823

FU Chang-zhen, ZAN Lin-sen, WANG Hong, JIANG Bi-jie, CHENG Gong,WANG Hong-bao, ZHU Guang-xing, LI Yao-kun, WANG Hong-cheng. Construction of Recombinant Adenovirus Vector Specific to SREBP1 Gene of Qinchuan Cattle and the Packaging and Amplifying of the Corresponding Adenovirus[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2013, 44(8): 1323-1329.

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Construction of Recombinant Adenovirus Vector Specific to SREBP1 Gene of Qinchuan Cattle and the Packaging and Amplifying of the Corresponding Adenovirus

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