一种新的人类G6PD突变型的体外构建

Construction of a new mutant of human G6PD in vitro

目的:体外构建人类G6PD突变型。方法:①运用PCR方法获得含有G6PD基因开放阅读框的PCR产物,随后将PCR产物连接到18T-simple vector中,构建成18T-G6PD;②酶切下G6PD cDNA的开放阅读框并连接到pALTER-1 vector中,构建成pAL-G6PD重组子;③运用含有突变序列的寡核苷酸引物体外构建G6PD835-海口突变重组子(835A→G,T279A),命名为pAL-G6PD-AG。结果:获得了G6PD的T279A的突变重组子。结论:此项工作为进一步体外表达突变型G6PD,并展开比较人类G6PD新发点突变835-海口(835A→G,T279A)与正常G6PD的酶动力学差异的研究奠定基础。

Objective： To construct a new mutant of human GSPD in vitro. Methods： （1）A normal G6PD cDNA open reading frame（ORF） was obtained by PCR from the GP plasmid （the plasmid of G6PD cDNA） and inserted into the 1ST-simple vector （named 18T-G6PD） ;（2）Then the normal G6PD cDNA ORF was cut down from the 18T-G6PD and inserted into the pALTER-1 vector （named pAL-G6PD）;（3）The synthetic oligonucleotide was used for site-directed mutagenesis. The pAL-G6PD-AG contained the mutant of 835-haikou （835A→G, T279A）. Results： The result showed that T279A mutant of G6PD was obtained. Conclusion： This work makes the base for further comparing the difference of the kinetics of enzyme-catalyzed reaction between the normal GSPD and the 835-haikou G6PD in vitro.