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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 .
In order to study the anti-angiogenic activity of recombinant mouse canstatin N-terminal fragment, cDNA of mouse canstatin N was amplified by PCR and cloned into prokaryotic expression vector pET30a (+). The recombinant plasmid pET mCanN was constructed, The recombinant plasmid pET mCanN was transformed into E.coli BL21 (DE3), induced by IPTG, and detected by SDS PAGE and Western blotting.The results showed that the IPTG induced prokaryotic expression vector pET mCanN was expressed in Escherichia coli E . The expression of the canstatin N fragment in mice was about 18% of the total bacterial protein. The canstatin N fragment of mouse mainly existed in the form of inclusion bodies. The inclusion bodies were washed, lysed, The recombinant mouse canstatin N fragment with a purity of about 92% was obtained after purified by co-column chromatography and protein renaturation.Experimental results showed that the canstatin N-terminal fragment of mouse prokaryotic expression Can effectively inhibit the formation of chicken embryo neovasculature in a dose-dependent manner.