Construction of recombinant adenoviral vector overexpressing betatrophin
JIANG Dan1， XUE Hao-ying1， WANG Su1， YANG Qi-chao1， LIU Yuan-xin1， YANG Ling1， WANG Dong1，YIN Wei1， ZHUANG Ruo1，QIAN Wei-yun2， ZHAO Shuang-xia3， ZHOU Li-bin4， YUAN Guo-yue1
（1. Department of Endocrinology，the Affiliated Hospital of Jiangsu University，Zhenjiang Jiangsu 212001； 2. Department of Gerontology，the Affiliated 3th Hospital of Suzhou University，Changzhou Jiangsu 213001； 3. Department of Endocrinology，Shanghai Jiaotong University Affiliated 9th People′s Hospital，Shanghai 200011； 4. Department of Endocrinology，Affiliated Ruijin Hospital of Medical School，Shanghai Jiaotong University，Shanghai 200025， China)
Abstract：[Abstract]Objective: To construct and identify the recombinant adenoviral vector expressing mouse betatrophin gene by using gene homologous recombination and to provide an experimental tool for further studying the function of betatrophin. Methods: From the sequence of GenBank,betatrophin gene including of BamHⅠ/ AgeⅠrestriction enzyme cutting sites was obtained by chemical synthesis and was inserted into the GV314 vector(CMVMCS3FLAGSV40EGFP) carrying enhanced green fluorescent protein(EGFP) to construct shuttle plasmid pGV314betatrophin. The plasmid GV314betatrophin was linearized with BamHⅠ/ AgeⅠand transfered into adenovirus genomics plasmid pDC315 by using gene homologous recombination in vitro to construct recombinant adenovirus vector Adbetatrophin.The DNA of recombinant adenovirus vector was finally linearized and transfered into HEK293T cells to package recombinant adenovirus particles.Recombinant adenovirus particles were repeated amplification in 293T cells to check and identify supernatant of adenovirus by PCR, western blotting and sequencing technologies. Results: By application of PCR, western blotting and DNA sequencing, the positive recombinant adenovirus containing betatrophin gene could be transferred into HEK293T cells and were packaged successfully. Conclusion: The recombinant adenovirus vector carrying mouse betatrophin gene was successfully constructed
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2016, Vol. 26 
