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目的构建含有定点突变的人PRKAG2基因表达载体。方法首先应用Trizol法抽提健康人全血mRNA,通过实时聚合酶链反应(RT-PCR)克隆得到野生型人PRKAG2 cDNA;继而通过聚合酶链反应(PCR)定点突变的方法获得突变型人PRKAG2基因,命名为G100S,最后应用酶切连接的方法得到重组载体pEGFP-G100S。结果Trizol法提取人细胞mRNA,经RT-PCR合成cDNA第一条链,特异性引物PCR扩增得到PRKAG2基因片段,目的片段条带在1 761 bp左右。分段克隆得到的G100S片段分别为300 bp与1 500 bp左右。转化Top10感受态细菌,PCR筛选阳性菌落5个克隆有3个克隆可见1 800 bp左右的阳性片段,筛选得到pEGFP-G100S重组阳性克隆。测序证实重组载体pEGFP-G100S构建成功。结论构建的含定点突变人PRKAG2基因重组载体pEGFP-G100S为进一步研究基因PRKAG2 G100S突变的功能奠定了基础。
Objective To construct a human PRKAG2 gene expression vector containing site-directed mutagenesis. Methods Human whole blood mRNA was extracted by Trizol method and wild-type human PRKAG2 cDNA was cloned by real-time polymerase chain reaction (RT-PCR). Then the mutant human PRKAG2 was obtained by polymerase chain reaction (PCR) site-directed mutagenesis The gene was named as G100S. Finally, the recombinant vector pEGFP-G100S was obtained by restriction enzyme digestion. Results The Trizol method was used to extract mRNA of human cells. The first strand of cDNA was synthesized by RT-PCR. The PRKAG2 gene fragment was amplified by PCR with specific primers. The band of the target fragment was about 1 761 bp. The fragment of G100S cloned was 300 bp and 1 500 bp, respectively. Top10 competent bacteria were transformed into the positive colonies of PCR-positive clones. Three clones showed positive clones of 1 800 bp, and the recombinant positive clones of pEGFP-G100S were screened. Sequencing confirmed that the recombinant vector pEGFP-G100S was successfully constructed. Conclusion The recombinant plasmid pEGFP-G100S containing the site-directed mutated human PRKAG2 gene laid the foundation for further study on the function of the PRKAG2 G100S mutation.