B7-1转基因细胞株的表达和检测

Expression and Detection of Human B7-1 Transgene Cell Line

目的构建B7-1基因真核表达载体并导入CAK-1肾癌细胞表达,为进一步研究基因的功能做材料准备。方法采用逆转录-聚合酶链反应(reverse transcription polymerase chain reaction,RT-PCR)方法,以正常肝脏组织cDNA为模板,扩增B7-1基因全长读码框架,并构建到真核表达载体pCDNA3.1中,将其导入CAK-1肾细胞瘤细胞,对转染后的细胞进行RT-PCR和Western blot分析。结果克隆测序结果证实,B7-1读码框架被成功插入真核表达载体pCDNA3.1的相应位点,与pCDNA3空载体转染对照细胞相比,pCDNA3-B-7载体转染的CAK-1细胞中B-7基因mRNA水平和蛋白水平均有明显升高。结论 B7-1转基因细胞株的成功构建和检测,为深入研究B7-1蛋白的生物学功能奠定了材料基础。

Objective To construct B7-1 eukaryotic expression vector and import CAK-1 expression in renal carcinoma cells and to prepare materials for further study of gene function.Methods By reverse transcription polymerase chain reaction（RT-PCR） method,normal liver tissue cDNA was utilized as template,B7-1 gene full-length reading frame was amplified,the eukaryotic expression vector pCDNA3.1 was constructed and imported to CAK-1 renal carcinoma cells.The transfected cells were identified by RT-PCR and Western blot analysis.Results The cloning and sequencing results demonstrated that B7-1 reading frame was successfully inserted the corresponding site of eukaryotic expression vector pCDNA3.1.Compared with pCDNA3 empty vector control cells,the B-7 mRNA and protein levels were significantly higher in the pCDNA3-B-7 vector-transfected cells.Conclusion Successful construction and detection of human B7-1 gene eukaryotic expression vector provides the ideal material for further study of the functional role of B7-1 protein in tumorigenesis.