维生素D受体野生型及FokⅠ突变型真核表达载体的构建

摘要

Objective To clone human vitamin D receptor gene (VDR) cDNA and construct the wild and Fok I mutant eukaryotic expression vector pcDNA3.1 (-) B-myc/his VDR. Methods The hepatic tissue was disrupted and total RNA was extracted. Human VDR gene cDNA was amplified by using RT-PCR and then suhcloned into pcDNA3.1(-) B-myc/his empty vector with using the restrictive endonucleases EcoR I and Hind Ⅲ , The Fok I mutant VDR plasmids were constructed by site-directed mutant methods. Results The VDR RT-PCR product was consistent with theoretic value 1300 bp. The clones contained the amplified VDR target gene segment were divided into two hands hy digestion of the restrictive endonucleases , one was 1300 bp corresponding VDR gene, the other was 5500 bp corresponding pcDNA3.1 (-) B-myc/his vector. The sequencing results showed that the sequence of human VDR gene from hepatic of Chinese was the same as that of human VDR gene in Genebank. Conclusions The plasmids for encoding hVDR cDNA were successfully cloned and their eukaryotic expression vector (wild type and FokI mutant) pcDNA3.1 (-) B-myc:/his hVDR were successfully constructed. The experiment foundation is establish successfully to further study the VDR gene variation and molecule mechanism of related tumor susceptibility.%目的:克隆人维生素D受体(VDR)全长,构建VDR野生型及FokⅠ突变型重组真核表达质粒.方法:提取总RNA,RT-PCR方法扩增将产物用EcoRⅠ和Hind Ⅲ进行双酶切后与pcDNA3.1(-) B-myc/his载体连接形成重组载体;对野生型VDR基因行定点诱变,构建突变型FokⅠ VDR质粒.结果:重组质粒被酶切为两条带,一条为1 300 bp(代表人VDR),一条为5 500 bp(空载体);片段大小与理论值相符.测序结果与Genbank序列完全相同.结论:成功克隆了人VDR基因全长并成功构建了人野生型和突变型FokⅠVDR重组真核表达载体pcDNA3.1(-)B-myc/his hVDR,为进一步研究VDR基因变异与相关肿瘤易感性的分子机制奠定了实验基础.