Betatrophin重组腺病毒过表达载体的构建

［摘要］目的: 应用基因同源重组技术，构建及鉴定携带小鼠betatrophin基因Gm6484(NM_001080940)的复制缺陷型重组腺病毒载体，为进一步研究betatrophin的功能奠定基础。方法: 根据基因库中登录的小鼠betatrophin基因Gm6484(NM_001080940)序列，经化学合成得到含BamHⅠ/ AgeⅠ酶切位点的小鼠betatrophin基因全长cDNA质粒，将其酶切后插入到带有增强型绿色荧光蛋白（enhanced green fluorescent protein，EGFP）基因的GV314载体CMV MCS 3FLAG SV40 EGFP中，得到重组病毒穿梭质粒pGV314 betatrophin；经BamHⅠ/ AgeⅠ双酶切后，与线性化的pDC315病毒基因组质粒体外同源重组，构建含有目的基因的腺病毒载体Ad betatrophin，酶切线性化重组腺病毒质粒后转染HEK293T细胞包装成重组病毒颗粒，经在HEK293T细胞反复扩增数代后，利用聚合酶链式反应（PCR）、蛋白质印迹法及测序鉴定重组的腺病毒。结果： 阳性克隆质粒Ad betatrophin在HEK293T细胞中成功包装出重组病毒，经PCR、蛋白质印迹及测序检测表明重组腺病毒包装成功。结论： 成功构建了携带小鼠betatrophin基因的复制缺陷型重组腺病毒过表达载体。

Construction of recombinant adenoviral vector overexpressing betatrophin

[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(CMV MCS 3FLAG SV40 EGFP) carrying enhanced green fluorescent protein(EGFP) to construct shuttle plasmid pGV314 betatrophin. The plasmid GV314 betatrophin was linearized with BamHⅠ/ AgeⅠand transfered into adenovirus genomics plasmid pDC315 by using gene homologous recombination in vitro to construct recombinant adenovirus vector Ad betatrophin.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