华支睾吸虫一个谷胱甘肽转移酶新基因的克隆与表达

Cloning and expression of a new gene of glutathione transferasefrom Clonorchis sinensis

目的 克隆华支睾吸虫(Clonorchis sinensis,Cs)谷胱甘肽转移酶(glutathione transferase,GST)的一个新基因,并在大肠杆菌中表达。方法 用PCR方法从华支睾吸虫成虫cDNA(质粒)文库中扩增新基因CsGST1,对DNA及其推导的氨基酸进行序列分析。将CsGST1克隆到原核表达载体pET-30a(+),在大肠杆菌BL21/DE3表达,用SDS-PAGE鉴定重组蛋白。结果 从华支睾吸虫成虫cDNA(质粒)文库扩增出642 bp的CsGSTl,序列分析显示它所编码的氨基酸序列与麝猫后睾吸虫GST的同源性最高(88％),并具有GST N-末端和C-末端的保守功能域。构建了重组质粒pET-30a(+)-CsGST1,SDS-PAGE显示CsGST1在大肠杆菌中得到了高效表达,pET-30a(+)-CsGST1的蛋白条带的分子量约为30 kDa。结论成功构建了CsGST1的原核表达载体,并在大肠杆菌中得到了高效表达,为进一步研究该基因的功能打下了基础。

To clone and express the new gene for glutathione transferase (GST) of Clonirchis sinesis.The gene CsGSTl was obtained from cDNA library (plasmid) of adult C. sinesis, and the DNA and deduced amino acid sequences were analyzed This gene was then cloned into the prokaryotic expression vector pET-30a( + ), and expressed in E. coli BL21/DE3. The recombinant protein was analyzed by SDS-PAGE. It was found that a DNA fragment encoding GST of 642 bp in length was obtained from the cDNA library of adult C. sinesis. Its deduced protein shared the highest identity with GST of Opisthorchis viverrini( 88 % ), and this protein was showed to have two conserved domains: the N-terminal and C-terminal domain of GST. In addition the recombinant plasmid pET-3a( + )-CsGSTl was constructed and the CsGST-1 gene was highly expressed in E. coli with a molecular size of 30 kDa of protein band as shown in SDS-PAGE analysis. It indicates that the prokaryotic expression vector of CsGST1 gene was successfully constructed, and highly expressed in E. coli . These can provide for a basis to study its functions.