TSLC1真核表达载体的构建及在HepG2肝癌细胞中的表达  

Construction of an eukaryotic expressing vector pReceiver-M29-TSLC1 and its expression in hepatoma HepG2 cells

作　　者：肖钟迪[1,2] 张玉成[3] 房学东[1] 

机构地区：[1]吉林大学第二临床医院,长春130041 [2]大庆油田总医院普外科,大庆163001 [3]吉林大学中日联谊医院中心研究室,长春130033

出　　处：《中国体视学与图像分析》2010年第1期89-94,共6页Chinese Journal of Stereology and Image Analysis

基　　金：吉林省发展与改革委员会资助课题[(2006)1550号]

摘　　要：目的构建TSLC1真核表达载体,并转染到肝癌细胞HepG2中使之表达,为研究TSLC1的抗肝癌作用奠定基础。方法利用RT-PCR技术扩增TSLC1基因全长,并与真核表达载体pRe-ceiver-M29进行连接;应用双酶切、PCR以及测序鉴定此连接载体;脂质体Lipofectamine 2000介导重组载体转染到HepG2细胞中,经G418筛选建立稳定转染细胞株,分别采用RT-PCR和免疫组化法检测其表达。结果RT-PCR获得了约1 329 bp大小的TSLC1基因片段;经过双酶切、PCR以及测序鉴定证实TSLC1基因片段正确插入真核表达载体pReceiver-M29中;与对照组比较,RT-PCR方法可见显示转染组细胞TSLC1mRNA表达明显增多,免疫组化法可见显示转染组细胞TSLC1蛋白表达明显增高。结论成功构建了真核表达载体pReceiver-M29-TSLC1,建立了稳定转染TSLC1的HepG2细胞株。Objective To construct an eukaryotic expressing vector pReceiver-M29-TSLC1 and examine its expression in hepatoma HepG2 cells.Methods The full length of TSLC1 was amplified and the pReceiver-M29-TSLC1 was constructed by polymerase chain reaction（PCR）.The pReceiver-M29-TSLC1 was identification using enzyme cutting,PCR and sequencing.After the pReceiver-M29-TSLC1 was transfected into hepatoma HepG2 cell mediated with Lipofectamine 2000,a stable transfected cell line was established using G418 screening and the TSLC1 expression was detected by RT-PCR and immunochemistry.Results The TSLC1 gene fragment was successfully obtained by PCR.The cDNA fragment of TSLC1 was accurately inserted into the vector of pReceiver-M29.Compared with the control group,TSLC1 mRNA expression was significantly increased in the transfected cell group identified by PR-PCR,and the TSLC1 protein expression was also higher in the transfected cell group detected by immunochemistry.Conclusion An eukaryotic expression vector pReceiver-M29-TSLC1 has been successfully constructed in this study and a stable transfected HepG2 cell line with TSLC1 established.

关 键 词：TSLC1 聚合酶链反应 克隆 HEPG2 

分 类 号：Q78[生物学—分子生物学]