固醇携带蛋白2腺病毒载体的构建与鉴定

Expression of Mouse SCP2 Gene Adenoviral Vector Carrying Albumin Promoter in Hepa1-6 Cells

目的:构建携带白蛋白启动子SCP2基因腺病毒载体,研究其与胆固醇结石形成的关系。方法:(1)利用RT-PCR技术克隆小鼠SCP2基因,在其上游接入白蛋白(ALB)启动子,下游连接绿色荧光报告基因(EGFP),构建穿梭质粒pDC312-ALB-SCP2-IRES2-EGFP;(2)采用Ad Max TM Adenoviru5 Vector系统包装病毒,CsCl法纯化病毒、TCID50法测定滴度;(3)重组腺病毒感染小鼠hepa-1-6细胞,实时定量PCR检测mRNA的表达;Western印迹检测SCP2蛋白表达情况;结果:成功构建携带白蛋白启动子SCP2基因腺病毒载体;当SCP2基因过表达时,CYP7a1基因表达下调,(t=3.97,P<0.05)HMGCR基因基因表达上调,(t=3.23,P<0.05)。结论:当SCP2基因过表达时引起HMGCR、CYP7a1基因转录水平的变化,提示SCP2基因可能通过影响胆固醇和胆汁酸的代谢而参与胆固醇结石的形成。

Objectives To construct the replication defective adenoviral vector of SCP2 gene carrying mu- fine albumin promoter, and study the relations between SCP2 gene and the formation of cholesterol calculus. Methods The cDNA of SCP2 gene was cloned by using RT-PCR technique. The albumin promoter was linked to SCP2 gene＇s upstream, and the EGFP gene lied in its downstream. The plasmid pDC312-ALB-SCP2-IRES2 -EGFP was constructed by the gene recombination technique. The Admax Adenoviral Vector System was used to generate the replication defective adenoviral vectors, which were purified by CsC1 method. The processes of TCID50 were applied to detect the titers of the adenoviral vectors. The RNA and protein were respectively ex- tracted from the infected Hepal-6 cells by the adenoviral vector. The real-time quantitative PCR was employed to detect the mRNA expression levels, and the Western blotting analysis was used to measure the SCP2 protein levels. Result We constructed successfully the replication defective adenoviral vector of SCP2 gene carrying murine albumin promoter. When the mRNA levels of SCP2 gene were overexpressed, CYP7al mRNA levels were down-regulated （t=3.97, P 〈 0.05）; and the mRNA levels of HMGCR were up-regulated （t=3.23, P 〈 0.05）. Conclusions The SCP2 gene overexpression may affect cholesterol and bile acid metabolism, which could promote the formation of cholesterol calculus.