携带人脂联素基因重组腺病毒的构建及表达

Construction and Expression of a Recombinant Adenovirus Carrying Human APM1 Gene

目的构建携带人脂联素基因的重组腺病毒载体,为进一步研究脂联素的功能提供实验基础。方法以带有人脂联素基因的质粒pINCY-APM1为模板,聚合酶链反应扩增人脂联素基因APM1,并将其定向克隆于真核表达载体pDC315-EGFP,与辅助质粒pBHGlox?E1,3Cre共转染HEK293细胞,经位点特异重组包装得到重组腺病毒Ad-APM1。通过Real-timePCR和Westernblot分别检测重组腺病毒Ad-APM1感染HEK293细胞后的表达。结果重组腺病毒Ad-APM1包装成功,病毒滴度>6.3×1012pfu/mL。Real-timePCR和Westernblot检测证实APM1在HEK293中表达。结论应用细胞内同源重组方法成功构建了携带人脂联素基因的重组腺病毒。

Objective To construct a recombinant adenovirus vector carrying human APM1 gene and provide experimental basis for further investigating on the function of adiponectin. Methods Human adiponectin gene was amplified by polymerase chain reaction （PCR） from plasmid pINCY-APM1 and cloned into eukaryotic expression vector pDC315-EGFP, then the products were co-transfected into HEK293 ceil line with adenovirus DNA helper plasmid pBHGlox △ E1, 3cre. The recombinant adenovirus Ad-APM1 was obtained by site-specific recombination. The expression of adiponectin was detected by Real-time PCR and Western blot respectively. Results The recombinant adenovirus-APM1 was constructed successfully with a titer higher than 6. 3× 10^12 pfu/mL. The expression of adiponectin in HEK293 ceils can be detected by Real-time PCR and Western blot. Conclusion The recombinant adenovirus carrying human APM1 gene was successfully constructed by the method of homologous recombination in cells.