人类G6PD顺德型〔Gd（－）Shunde〕基因在大肠杆菌中的表达

Expression of a human glucose-6-phosphate dehydrogenase mutant gene,Gd(-)Shunde,in E. Coli

为了证明一种人类新突变型Ｇ６ＰＤ顺德型，ｃＤＮＡ５９２Ｃ→Ｔ与酶活性降低和其生化特性改变有关，将正常Ｇ６ＰＤｃＤＮＡ，插入人工诱变载体ｐＡＬＴＥＰＲ－１中，用体外定点诱变技术，将ｃＤＮＡ５９２位的Ｃ置换为Ｔ，然后再插入ｐＡＣＩ，在其本身无Ｇ６ＰＤ活性的大肠杆菌ＨＢ３５１突变株中进行表达。实验证明，从ＨＢ３５１株表达出的人Ｇ６ＰＤ，酶活性降低，ＫｍＧ６Ｐ降低，三种底物同类物利用率明显增高，与人类Ｇ６ＰＤ顺德型的生化参数完全一致，表明５９２Ｃ→Ｔ是引起Ｇ６ＰＤ活性降低和生化特性改变的直接原因。

To demonstrate the relationship between biochemical characteristic changesof the new G6PD variant Shunde and the mutation cDNA 592C-T, a normal G6PD cDNAwas cut down from plasmid pAC1 and inserted into a vector (pALTER-1).A syntheticol igonucleotide was used for site-directed mutagenesis.The G6PD cDNA with an inducedmutation 592c Twas then inserted into pAC1,and the mutated G6PD was cxpressed inE。 Coli HB351(a mutant which lost its own G6PD activity)。The results showed that the decreased enzyme activity,the low Km G6P, a nd the in-creased three substrate ana logue utilization rates were all in concordance with the human-G6PD Shunde.These results demonstrated directly that the cDNA592 C-T was the causeof biochemical changes of variant G6PD Shunde。