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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HindⅢ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FokⅠVDR质粒。结果:重组质粒被酶切为两条带,一条为1300bp(代表人VDR),一条为5500bp(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/hishVDR,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。
OBJECTIVE: To clone full-length human vitamin D receptor (VDR) and construct a recombinant eukaryotic expression vector of VDR wild-type and Fok I mutant. METHODS: The total RNA was extracted and amplified by RT-PCR. The product was double-digested with EcoRⅠand HindⅢ and ligated with pcDNA3.1 (-) B-myc / his vector to form a recombinant vector. The wild-type VDR gene was subjected to site-directed mutagenesis To construct a mutant Fok IVDR plasmid. Results: The recombinant plasmids were digested into two bands, one for 1300bp (representing human VDR) and the other for 5500bp (empty vector). The fragment size was consistent with the theoretical value. The sequencing result is exactly the same as the Genbank sequence. Conclusion: The full-length human VDR gene was successfully cloned and the recombinant eukaryotic expression vector pcDNA3.1 (-) B-myc / hishVDR of human wild-type and mutant FokⅠVDR was successfully constructed. To further study the relationship between VDR gene mutation and related tumor susceptibility The molecular mechanism has laid the experimental foundation.