钙网蛋白122～180片段基因克隆、表达和活性分析

Calreticulin 122～180 Fragment Inhibits Angiogenesis and Suppresses Tumor Growth

钙网蛋白是高等动物细胞中普遍存在的一种钙结合蛋白 ,近年发现它及其N端 1～ 180位氨基酸能抑制内皮细胞生长和血管生成 .为了寻找高效和小分子质量的血管生成抑制因子 ,用PCR技术扩增出钙网蛋白N端12 2～ 180位氨基酸的DNA序列 ,克隆进原核表达载体pET 3c ,转化大肠杆菌BL2 1(DE3) ,经IPTG诱导后 ,该片段以包涵体形式表达 ,表达量约占菌体总蛋白的 35 4 % .包涵体经变性溶解、复性和初步纯化后 ,纯化产物可以抑制人脐静脉内皮细胞的生长 ,鸡胚绒毛尿囊膜的血管生成和小鼠原位黑色素瘤的生长 .

Calreticulin is an calcium binding protein existed in all animal cells. It was found that calreticulin and its N terminal 1～180 amino acid inhibit the proliferation of endothelial cells and angiogenesis. In order to search for efficient and small molecular angiogenesis inhibitor, the DNA sequence coding for the N terminal 122～180 amino acid was amplified by PCR and cloned into the prokaryotic vector pET-3c. The recombinant plasmid pET-N58 was transformed into E.coli BL21(DE3). The recombinant protein was expressed efficiently in E.coli BL21(DE3,pET-N58) as inclusion body with a yield of about 35.4% of the bacterial total protein. The partial purified recombinant protein inhibits the proliferation of human umbelical vein endothelial cells and the CAM angiogenesis. The recombinant protein also suppresses the growth of primary B16 murine melanoma in C57BL/6 mice.