血管性血友病因子裂解酶金属蛋白酶域的克隆和表达  

Cloning and expression of the metalloproteinase domain of human von Willebrand factor-cleaving protease

作　　者：高维强[1] 白霞[1] 傅建新[1] 阮长耿[1] 

机构地区：[1]苏州大学附属第一医院,江苏省血液研究所江苏苏州215006

出　　处：《中国病理生理杂志》2004年第10期1832-1836,共5页Chinese Journal of Pathophysiology

摘　　要：目的 :克隆和表达血管性血友病因子裂解酶 (vWF -cp)的金属蛋白酶域。方法 :应用PCR从含有人vWF -cp金属蛋白酶域的质粒中扩增出该区域 ,克隆至pUC18载体后行序列分析 ,并将其重组于带有 6×HisTag的融合蛋白表达载体pET2 8a(+)中 ,在大肠杆菌DE3/plySs中诱导表达。融合蛋白经Ni-NTA柱纯化后 ,采用Westernblot印迹鉴定。结果 :PCR扩增得到 6 5 8bp的vWF -cp金属蛋白酶域的基因片段 ,测序结果与GenBank序列一致。重组表达质粒经异丙基硫代 - β -D -半乳糖苷 (IPTG)诱导 5h后表达的融合蛋白占菌体总蛋白的 2 8% ,进一步纯化后其纯度在 95 %以上。结论 :在大肠杆菌中高效表达了人vWF -cp的金属蛋白酶结构域 ,为深入研究vWFAIM: To clone and express the metalloproteinase domain of human von Willebrand factor-cleaving protease (vWF-cp). METHODS: The metalloproteinase domain of human vWF-cp, amplified from the plasmid containing the vWF-cp cDNA gene by using polymerase chain reaction, was cloned into pUC18, and its accuracy was verified by sequencing. Then the domain was inserted into the multiclone site of pET28a(+) and included a 6×His Tag at its amino terminal. After induced by IPTG, the recombinant protein was purified by using a Ni-NTA column and confirmed by Western blot. RESULTS: Comparison of the nucleotide sequence of our cloned domain with the GenBank sequence revealed no difference. High-level expression of the recombinant protein was yielded after 5-hour induction, which amounted to 28% of total bacteria protein in inclusion body. Western blot demonstrated that it possessed high specificity. CONCLUSION: The metalloproteinase domain of vWF-cp was high efficiently expressed in Escherichia coli. This might contribute to the further study of the relationship between its structure and function. [

关 键 词：von WILLEBRAND因子 克隆 基因 基因表达 

分 类 号：R363[医药卫生—病理学]