转染超抗原SEA肝癌细胞的构建及鉴定

Construction and Identification of the Superantigen SEA Transfected Hepatocellular Carcinoma Cells

目的用基因重组的方法将葡萄球菌肠毒素A(SEA)基因转染至肝癌细胞.方法先从产SEA标准菌株中提取并扩增SEA全长基因,将SEA基因克隆及亚克隆至真核表达载体pLXSN构建pLXSN-SEA重组质粒,再将重组质粒转染至人肝癌细胞系BEL-7402细胞,用RT-PCR和ELISA法鉴定.结果从产SEA标准菌株ATCC13565中提取并扩增出SEA基因全长片段;SEA基因克隆和亚克隆至真核表达载体pLXSN,经测序证实所得序列与GenBank中的标准序列完全一致;将pLXSN-SEA质粒转染肝癌细胞BEL-7402,经筛选获得稳定表达的抗性单克隆.RT-PCR扩增出约800bp的基因片段,经ELISA分析细胞培养上清中SEA蛋白的含量在皮克的水平.结论成功构建了重组超抗原SEA基因的克隆载体及真核表达载体,将SEA基因转染至肝癌细胞株BEL-7402细胞后癌细胞能够表达并持续分泌SEA蛋白.

Objective To use genetic recombination method to clone the superantigen Staphylococcal enterotoxin A （SEA） gene and transfect it to hepatocellular carcinoma cells. Methods SEA gene fragment was obtained from standard bacteria; The pLXSN - SEA recombinant plasmid was constructed and identified;then pLXSN - SEA recombinant plasmid was transfected to human hepatocellular carcinoma cells line BEL- 7402 cells and identified by RT- PCR and ELISA method. Results pLXSN - SEA had been constructed successfully and proved as same as the standard gene rank in Genbank;pLXSN - SEA recombinant plasmid was transfected to human BEL - 7402 cells and the concentration of SEA protein in supernatants was about pg level with ELISA method. Conclutions A cloning vector and eukaryon - expression vector of recombination superantigen SEA gene was successfully constructed, and the SEA gene was transfected to hepatocellular carcinoma cells line BEL - 7402 cells. This cell could express and excrete SEA protein continuesly.