人肝再生增强因子基因慢病毒表达载体的构建与鉴定  

Construction and Identification of Lentivirus Expression Vector Carrying Human ALR

作　　者：薛峰[1] 王伯庆[1] 尹继炜[1] 易超[1] 

机构地区：[1]新疆医科大学附属肿瘤医院肝胆胰外科,乌鲁木齐830011

出　　处：《成都医学院学报》2015年第4期393-396,409,共5页Journal of Chengdu Medical College

基　　金：中国高校医学期刊临床专项资金(No:11523021)

摘　　要：目的构建携带人肝再生增强因子(augmenter of liver regeneration,ALR)基因的慢病毒表达载体,并验证其安全性与有效性。方法将以人胎肝cDNA为模板扩增得到的ALR与pLenti6/V5-D-TOPO重组慢病毒载体连接后,与慢病毒包装体系ViraPowerTMPackaging Mix共转染293T细胞,得到携带人ALR基因的慢病毒表达载体病毒颗粒,实时定量PCR法测定病毒滴度;转染BLR 3A大鼠肝细胞,观察细胞生长情况,并用RT-PCR法测定ALR基因表达的方法,验证构建的慢病毒载体的安全性与有效性。结果扩增产物凝胶电泳、提抽质粒酶切电泳及基因测序结果均显示成功构建携带ALR基因的慢病毒表达载体,测得其病毒滴度为1.48×107 v.p./mL。转染BLR 3A大鼠肝细胞72h后,仅少量细胞病变脱离,转染率为88.49%;RT-PCR结果显示,构建的携带ALR慢病毒表达载体可诱导BLR 3A大鼠肝细胞表达ALR基因。结论慢病毒表达载体是一种兼具有效性与安全性,且适用于基因研究的病毒表达载体。Objective To construct lentivirus expression vector carrying human augment of liver regeneration （ALR） and to verify its efficacy and safety by transfecting hepatic cells of BLR 3A rats. Methods After being connected with pLenti6/V5-D- TOPO-recombinant lentivirus expression vector, ALR that was obtained by human fetal liver cDNA amplification was used to transfect 293T cells combining with lentivirus packaging system ViraPowerTM Packaging Mix to collect ALR-carrying lentivirus expression vector virion. Real-time （RT） quantitative polymerase chain reaction （PCR） method was used to detect the viral titer. Hepatic cells of BLR 3A rats were transfected to observe cellular growth,and RT-PCR was applied to detect the expression of ALR genes so as to verify the efficacy and safety of constructed lentivirus vector. Results The results of amplified product gel electrophoresis,enzyme digestion and polyacrylamide gel electrophoresis for extraction of plasmids as well as gene sequencing showed successful construction of ALR gene-carrying lentivirus expression vector, with viral titer of 1.48 × 107 v. p./mL obtained. After hepatic cells of BLR 3A rats were transfected for 72 h,only a few cells had lesions and isolation, with transfecting rate of 88. 49%. RT-PCR results indicated that the constructed ALR-carrying lentivirus expression vector could induce hepatic cells of BLR 3A rats expressing ALR genes. Conclusion Lentivirus expression vector is a safe and effective vector and is applicable for lentivirus expression vector in genetic research.

关 键 词：肝再生增强因子 慢病毒表达载体 BLR 3A 大鼠肝细胞 基因重组技术 

分 类 号：Q78[生物学—分子生物学]