hepaCAM基因重组腺病毒质粒的构建及鉴定  

Construction and identification of recombinant adenovirus vector for expression of hepaCAM gene

作　　者：陶佳[1] 王秋菊[1] 范砚茹[1] 杜红飞[1] 宋学东[1] 罗春丽[1] 吴小侯[2] 

机构地区：[1]重庆医科大学检验医学院临床检验诊断学教育部重点实验室,重庆400016 [2]重庆医科大学附属第一医院泌尿外科,重庆400016

出　　处：《中国生物制品学杂志》2013年第5期639-642,共4页Chinese Journal of Biologicals

基　　金：国家自然科学基金(81072086)

摘　　要：目的构建表达肝细胞黏附分子(Hepatocyte cell adhesion molecule,hepaCAM)基因的重组腺病毒质粒,并进行鉴定。方法以质粒pEGFP-N2-hepaCAM为模板,PCR扩增hepaCAM基因,克隆至腺病毒穿梭质粒pAdtrack-CMV上,经酶切、PCR及测序鉴定正确后,将重组腺病毒穿梭质粒pAdtrack-CMV-hepaCAM经PmeⅠ线性化,转化至含腺病毒骨架质粒pAdEasy-1的感受态大肠杆菌BJ5183中进行同源重组。重组腺病毒质粒pAdEasy-1-hepaCAM经PmeⅠ线性化后,转染HEK-293细胞,包装重组腺病毒AdI-hepaCAM,经大量扩增后,检测病毒滴度。RT-qPCR及Westernblot检测感染的BIU-87细胞内hepaCAM的表达。结果酶切鉴定证实重组腺病毒质粒pAdEasy-1-hepaCAM构建正确,重组腺病毒AdI-hepaCAM滴度可达1.6×1012pfu/ml,与空载体腺病毒组比较,经重组腺病毒感染的BIU-87细胞中hepaCAM mRNA及蛋白的表达均明显升高(P<0.05)。结论已成功构建携带hepaCAM的重组腺病毒载体AdI-hepaCAM,为研究hepaCAM基因对膀胱癌细胞增殖的调控提供依据。Objective To construct and identify the recombinant adenovirus vector for expression of hepatocyte cell adhesion molecule（hepaCAM）gene.Methods The hepaCAM gene fragment was amplified by PCR using plasmid pEGFPN2-hepaCAM as a template,and cloned into shuttle plasmid pAdtrack-CMV.The constructed recombinant shuttle plasmid pAdtrack-CMV-hepaCAM was linearized with PmeⅠ,and transformed to competent E.coli BJ5183 for homologous recombination with adenoviral backbone plasmid pAdeasy-1.The obtained recombinant adenovirus plasmid pAdEasy 1hepaCAM was linearized with Pac Ⅰ and transfected to HEK-293 cells for packaging.The obtained recombinant adenovirus AdI-hepaCAM was propagated in a large quantity and determined for titer.The expression of hepaCAM in BIU-87 cells infected with adenovirus was determined by RT-qPCR and Western blot.Results Restriction analysis proved that recombinant adenovirus plasmid pAdEasy-1-hepaCAM was constructed correctly.Recombinant adenovirus AdI-hepaCAM reached a titer of 1.6×10 12 pfu/ml.Both the mRNA transcription and protein expression levels of hepaCAM in BIU-87 cells infected with AdI-hepaCAM increased significantly as compared with those infected with AdI-EGFP（P〈0.05）. Conclusion The recombinant adenovirus vector for expression of hepaCAM gene was constructed successfully,which provided a basis for study on regulatory effect of hepaCAM gene on proliferation of bladder cancer cells.

关 键 词：黏附分子 腺病毒 膀胱癌 

分 类 号：Q782[生物学—分子生物学]