人vW因子A3区基因的克隆及其序列分析

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

Cloning of von Willebrand Factor-A3 Domain and Its Sequence Analysis

Objective von Willebrand factor (vWF) mediates platelet adhesion through binding to platelet glycoprotein Ib(GPIb) and extracellular matrix(ECM). Previous studies identified that vWF domain A1 was involved in binding GPIb and domain A3 related to binding to collagen. To further investage the mechanism of thrombus formation and develop a new remedy of anti-thrombus formation. Methods RNA was extracted from human umbilical vein endothelial cells, RT-PCR was adopted to amplify DNA of vWF domain A3 , the target gene sequence was analysed by auto-analyser of DNA sequence.Results The target gene was amplified from human umbilical vein endothelial cells through RT-PCR method, and it was inserted into pUCmT vector at the presence of T4 DNA ligase, its sequence was identified to comform with published vWF domain A3.Conclusion DNA of vWF domain A3 is amplifed and recombinant containing target gene constructed successfully. The above experiments will lay a foundation in expression of its protein and research of its function