pLNCX-pemt2重组载体的构建及pemt2基因在大鼠肝癌CBRH-7919细胞中的表达

Construction of pLNCX-pemt2 Vector and Expression of pemt2 Gene in Hepatoma CBRH-7919 Cells of Rats

为进一步研究 pemt2对肝癌细胞生长抑制的作用机制提供方便的实验模型 ,构建了p LNCX- pemt2重组体 .将目的基因 pemt2连接入含有 neo抗性基因的真核细胞表达载体 p LNCX中 ,构建 p LNCX- pemt2重组子 ,并用磷酸钙沉淀法将其转入大鼠肝癌 CBRH- 791 9细胞中 ,应用PCR、Western印迹及 [3 H]SAM参入等技术对其转染、表达及活性进行鉴定 .转染 p LNCX- pemt2的大鼠肝癌细胞 ,PEMT2成功表达 (分子量为 2 2 .5k D) ;高表达克隆 PEMT2的表达量比对照组高约 5倍 ,其活性比对照组高 2 .1倍 ;细胞生长的倍增时间从 2 1 .54± 7.0 8h延长到 43.2 2± 7.1 1h.结果表明 ,p LNCX- pemt2重组体转入肝癌细胞后 ,PEMT2蛋白得到高效表达 ,明显抑制肝癌细胞生长 .

To construct a plasmid carrying pemt2 and neo resistant genes for the mechanistic study of growth inhibition of pemt2 transfected hepatoma cells,pLNCX pemt2 vector with neo resistant gene was constructed,and transfected into CBRH 7919 hepatoma cells by calcium phosphate precipitation method.PCR,Western blot and [ 3H]SAM incorporation were used for checking transfection and for activity determination,respectively.An overexpression clone of PEMT2 was screened out in which the enzyme quantity was about 5 fold,and the enzyme activity was 2.1 fold higher than those of the control.The expression product had an M r of 22.5 kD.The cell generation time of the overxpression Clone was 43.22±7.11 h,but the control one was just 21.54±7.08 h.The results showed that the pLNCX pemt2 was successfully constructed and transfected into rat hepatoma cells,and a clone with over expression of PEMT2 was established.The growth of this clone was significantly retarded.