人G6PD T279A和T279S两种突变子的体外构建

Construction of T279A and T279S Gene Mutants of Glucose 6-Phosphate Dehydrogenase

目的:构建葡萄糖6磷酸脱氢酶(Glucose 6-phosphate dehydrogenase,G6PD)T279A和T279S两种突变子。方法:以Genbank No X03674为参考序列设计并合成引物、以含G6PD基因的质粒(Philip J Mason博士惠赠)为模板,PCR扩增获得G6PD野生型基因片段,琼脂糖凝胶电泳后回收PCR产物,连接、转化构建克隆质粒pMD18T-G6PD;酶切pMD18T-G6PD质粒、电泳后回收目的基因片段,连接、转化构建含G6PD野生型基因的重组质粒pAL-G6PD;设计并合成含有突变序列的引物,以pAL-G6PD为模板,体外扩增获得G6PD835-海口(835A→T279A)和835-中国-1(835 A→T,T279S)突变子。结果:酶切后经电泳鉴定表明获得与预期大小相符的pMD18T-G6PD质粒,EcoRⅠ和HindⅢ双酶切获得与预期大小相符的pAL-G6PD,测序结果与参考序列完全一致。0.8%的琼脂糖凝胶电泳鉴定,并定量pAL-G6PD单链DNA浓度约为200ng/uL。经测序鉴定并与参考序列比对结果表明获得了G6PD的T279A和T279S两种突变子。结论:成功构建了G6PD的T279A和T279S两种突变子,为下一步原核表达、生化性质以及酶动力学等研究奠定了基础。

Objective： To construct the T279A and T279S gene mutants of Glucose 6-phosphate dehydrogenase （G6PD）. Method： According to reference sequence （Genbank No X03674 ）, the gene fragment of G6PD was amplified from a plasmid containing G6PD gene gifted by Dr. Philip J Mason. This gene fragment was inserted into pMDl 8-T-simple vector to get a plasmid pMDl 8T-G6PD, which was digested by restriction enzyme and the products were recovered and inserted into pALTER-1 for pAL-G6PD. Subsequently, oligonucleothides containing mutation sequence were designed and synthesized, and polymerase chain reaction （PCR） was conducted for the pAL-G6PD-AG containing the mutant of 835-Haikou （835A→G,T279A） and the pAL-G6PD-AT contained the mutant of 835-chinese-1 （835A→T,T279S） using pAL-G6PD as template. Results： The matched pMDl 8T-G6PD and pAL-G6PD plasmids were obtained and confirmed by restriction enzyme analysis. And the sequenced result of pAL-G6PD was consistent with the reference sequence. By 0.8% agarose gel electrophoresis, the density of single strand ofpAL-G6PD was 200ng/uL. comparing with the reference sequences, the two mutants of T279A and T279S were confirmed to be constructed successfully. Conclusions： Two mutants of T279A and T279S containing G6PD have been constructed successfully, which provide a tool for futher study on prokaryotic expression, biochemistry and enzyme kinetics.