突变型人PRKAG2基因的克隆及其真核表达载体构建

Cloning and the expression vector construction of mutant human PRKAG2 gene

目的构建含有突变位点R302Q的PRKAG2的真核表达载体。方法首先通过RT-PCR获得人PRKAG2的基因序列,然后采用PCR定点突变获得含有R302Q的PRKAG2基因序列,最后通过酶切连接至真核表达载体pIRES2-EGFP中。结果通过RT-PCR获得的人PRKAG2的编码区序列1710 bp,经测序比对与NCBI公布的序列完全一致,通过PCR定点突变拼接900 bp和800 bp片段获得了含有R302Q突变位点的PRKAG2,酶切连接至pIRES2-EGFP载体中,经菌落PCR筛选和酶切验证获得阳性克隆pR302Q-IRES2-EGFP,经测序验证与预期完全一致。结论构建含定点突变人PRKAG2基因重组载体pR302Q-IRES-EGFP为进一步研究基因PRKAG2 R302Q突变体的功能奠定了基础。

Objective To construct human PRKAG2 gene expression vector containing site-directed mutagenesis. Methods First, gene sequence of human PRKAG2 was obtained through the access of real-time polymerase chain reaction （RT-PCR） from human whole blood mRNA, then, gene sequence of human PRKAG2 containing R302Q was also obtained through PCR and site-directed mutagenesis of human PRKAG2. Finally, the recombinant vector pR302Q-IRES2-EGFP was constructed by digested connection. Results The human cell mRNA was extracted with the Trizol method, and the first strand cDNA was synthesized with specific primers, the PRK- AG2 gene fragment was amplified by RT-PCR, and the target fragment bands were about 1710 bp. The R302Q fragments obtained by segment cloned were about 800 bp and 900 bp. After the Top 10 competent bacteria were transformed, positive bacteria were screened by PCR, further identified by restriction analysis. It was confirmed that the recombinant vector pR302Q-IRES2-EGFP was successfully constructed and was as fully consistent as predicted by sequencing. Conclusion The construction of recombinant vector pR302Q- IRES2-EGFP containing site-directed mutagenesis of human PRKAG2 gene lays a good foundation for further research on the function of the mutant PRKAG2 R302Q.