人canstatin基因的克隆、表达、纯化及其生物活性测定

目的:克隆人Canstatin cDNA,在E.coli中融合表达和纯化,并测定表达产物抑制新生血管生成活性.方法:从中国人胎盘组织提取总RNA,RT-PCR扩增出Canstatin cDNA,克隆入pTYB1载体中并测序,构建原核表达载体pTYB1-hCAN,IPTG诱导表达,几丁质亲和纯化,鸡胚绒毛尿囊膜(CAM)血管生成实验进行活性测定.结果:RT-PCR产物约为700bp,测序结果与文献报道序列一致.构建了人Canstatin原核表达载体vTYB1-hCAN,在大肠杆菌中表达.纯化的融合蛋白在CAM实验中能明显抑制血管生成.结论:克隆、表达了具有生物学活性的人canstatin蛋白.

Cloning, expression and purification of human canstatin and assay of its bioactivity

Objective:To clone human canstatin cDNA,express it as fusion protein in E.coli,purify the fusion protein and determine its biological activity.Methods:Total RNA was extracted from Chinese placenta tissue.The canstatin cDNA was amplified by RT-PCR,then cloned into pTYB1 and sequenced;Prokaryotic expression vector pTYB1-hCAN was constructed;fusion protein was expressed by using IPTG induction and purified by chitin column.The biological activity of the purified fusion protein was identified by using the chick embryo chorioallantoic membrane assay(CAM).Results:RT-PCR product was about 700 bp,its sequence was the same as that of canstatin reported.Prokaryotic expression vector pTYB1-hCAN of human canstatin was constructed successfully,and the fusion canstatin protein was expressed in E.coli.The purity of the protein was 91.6%.The fusion protein could suppress the angiogenesis in CAM.Conclusion:The active human Canstatin is cloned and expressed as a bioactive form.