云南阿昌族G6PD基因突变G487A在DF213中的表达

EXPRESSION OF G6PD GENE G487A MUTATION IN DF213 FROM ACHANG PEOPLE OF YUNNAN

为获得阿昌族G6PDWT和G6PDG487A重组蛋白,研究G6PDG487A的结构和功能改变,从云南省德宏州梁河县杞木寨乡湾中村阿昌族聚集地的G6PD缺陷家系先证者和正常阿昌族个体全血提取RNA,经RT-巢式PCR得cDNA,将cDNA克隆至pMD18-Tsimple载体中并测序;错配碱基经定点突变修复后,目的基因亚克隆至pThioHis(A)载体,构建了阿昌族G6PD基因野生型和G487A突变型原核表达载体:pThioHis(A)-AChang-G6PDWT和pThioHis(A)-AChang-G6PDG487A。用重组质粒转化E.coli Competent Cells DF213(G6PD defeciency),经IPTG诱导G6PD表达、10%SDS-PAGE电泳检测表达蛋白和紫外340nm定量测定G6PD活性的分析表明,pThioHis(A)-AChang-G6PDWT和pThioHis(A)-AChang-G6PDG487A在DF213中成功表达,分子量约为59kDa。IPTG诱导0、3、6、9、和12h后,G6PD活性逐渐增高,G6PD基因WT表达的酶活性约是G487A的20-25倍。表达载体的构建以及G6PDcDNA在DF213中成功表达,为重组酶G6PDG487A的进一步研究奠定了基础。

In order to get the recombinant protein of G6PD^WT and G6PD^G487A and study the changes on the structure and function of G6PD^G487Ain AChang People, full-length cDNA coding human G6PD gene was obtained by RT-nest PCR from the proband of G6PD deficiency and normal subject in AChang people. G6PD cDNAs were cloned into pMD18-T simple vector and the mismatch bases were corrected by using the site-mutation technique; Then cDNA were sub-cloned into the expression vector pThioHis （A） and expressed in E.coli DF213 （G6PD deficiency）. 10% SDS-PAGE electrophoresis analysis showed that the expressional G6PD native protein molecular mass was about 59 kDa. G6PD activity increased gradually after 0, 3, 6, 9 and 12 hours of IPTG induction by monitoring the rate of reduction of NADP＋ to NADPH at 340 nm spectrophotometrically. G6PD^WT activity was about 20-25 times of G6PD^G487A activity. In conclusion, the prokaryotic expressional vectors, pThioHis（A）-AChang-G6PD^WT and pThioHis（A）- AChang-G6PD^G487A, are constructed and expressed in DF213 successfully. It will be helpful for further study of recombinant enzyme with G6PD^G487A.