【摘要】 为了研究重组小鼠canstatinN端片段的体内抗血管生成活性 ,通过PCR扩增小鼠canstatinN端片段cDNA ,定向克隆于原核表达载体 pET30a (+)中 ,构建小鼠canstatinN端片段重组表达载体 pET mCanN ,转化E .coliBL2 1(DE3) ,IPTG诱导表达 ,SDS 聚丙烯酰胺凝胶电泳 (SDS PAGE)和蛋白质印迹检测小鼠canstatinN端片段的表达 .结果表明 ,IPTG诱导原核表达载体 pET mCanN在大肠杆菌E .coliBL2 1(DE3)中高效表达 ,小鼠canstatinN端片段表达量约占菌体总蛋白量的 18% ,小鼠canstatinN端片段主要以包涵体形式存在 ,包涵体经过洗涤、裂解、Ni spincolumn亲合柱层析以及蛋白质复性等步骤纯化后 ,获得了纯度约为 92 %的重组小鼠canstatinN端片段 .鸡胚绒毛尿囊膜 (chickenembryochoriollantoicmembrane ,CAM)实验表明 ,原核表达的小鼠canstatinN端片段能有效地按剂量依赖的方式抑制鸡胚新生血管的形成 .更多还原

【Abstract】 Mouse canstatin is a C-terminal globular non-collagenous domain of type Ⅳ collagen α2 chain, which previously showed anti-angiogenic activity. To investigate in vivo angiostatic effects on chick chorioallantoic membrane(CAM) by recombinant mouse canstatin N-fragment(1～95aa), the cDNA for N-fragment of mouse canstatin, obtained from a cloning vector pMD18T-mCan by PCR, was introduced into an expression vector pET30a(+) to construct prokaryotic expression vector pET-mCanN. N-fragment of mouse canstatin efficiently expressed in E.coli BL21(DE3) after IPTG induction was monitored by SDS-PAGE and by Western blotting with an anti-hexahistidine tag antibody. The expressed N-fragment of mouse canstatin, mainly as inclusion bodies, accounted for approximately 18% of the total bacterial proteins as estimated by densitometry. The inclusion bodies were washed, lysed and purified by the nickel affinity chromatography to a purity of 92%. The refolded N-fragment of mouse canstatin was tested on the chicken embryo CAMs, and a large number of newly formed blood vessels were significantly regressed. It is concluded that N-fragment derived from mouse canstatin is an effective inhibitor of angiogenesis of the chicken embryo in a dose-dependent manner through CAM assay and a promising candidate for the treatment of cancer, suggesting that these novel findings may add to the understanding of anti-angiogenesis effects of the N-fragment of mouse canstatin.更多还原