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目的 克隆人血管性血友病因子 (vonWillebrandFactor,vWF)A3区基因并进行序列分析。方法 应用Trizol一步法从原代培养的人脐静脉内皮细胞中抽提总RNA ,应用RT -PCR扩增vWFA3区基因 ,并与 pUCmT载体连接构建成重组克隆载体pUCmT -vWF -A3 ,全自动测序仪测定目的基因DNA序列。结果 RT -PCR从内皮细胞中扩增出约 60 0bp的DNA片段 ,酶切鉴定插入目的基因片段的大小与理论计算一致 ,重组克隆载体pUCmT -vWF -A3经测序其结果完全正确。结论 本法可成功地克隆人vWFA3区基因片段 ,为进一步研究其生物学功能奠定了基础
Objective To clone the gene of A3 region of von Willebrand Factor (vWF) and analyze its sequence. Methods Total RNA was extracted from primary cultured human umbilical vein endothelial cells by Trizol one-step method. The vWFA3 gene was amplified by RT-PCR and ligated with pUCmT vector to construct recombinant cloning vector pUCmT-vWF-A3. The instrument determines the DNA sequence of interest. Results RT-PCR was used to amplify a DNA fragment of about 60 bp from endothelial cells. The size of the inserted fragment was the same as that of the theoretical calculation. The result of sequencing of the recombinant cloning vector pUCmT-vWF-A3 was completely correct. Conclusion This method can be successfully cloned human vWFA3 gene fragment, which laid the foundation for further study of its biological function