重组小鼠canstatin N端片段对鸡胚新生血管生成的抑制作用(英)

为了研究重组小鼠canstatin N端片段的体内抗血管生成活性, 通过PCR扩增小鼠canstatin N端片段cDNA，定向克隆于原核表达载体pET30a(+)中,构建小鼠canstatin N端片段重组表达载体pET-mCanN, 转化E.coli BL21(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canstatin N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.coli BL21(DE3)中高效表达, 小鼠canstatin N端片段表达量约占菌体总蛋白量的18%, 小鼠canstatin N端片段主要以包涵体形式存在,包涵体经过洗涤、裂解、Ni-spin column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canstatin N端片段. 鸡胚绒毛尿囊膜(chicken embryo choriollantoic membrane,CAM)实验表明，原核表达的小鼠canstatin N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.

Angiostatic Effects on Chick Chorioallantoic Membrane by Recombinant N-Fragment of Mouse Canstatin

Mouse canstatin is a C-terminal globular non-collagenous domain of type 1V 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.