重组人canstatin的克隆、表达及其活性鉴定

目的: 克隆并表达canstatin基因，初步检测其抑制血管生成的活性。方法: 采用RTPCR方法从人胚肝组织中钓取canstatin cDNA，克隆入pMD18T载体中，并测序鉴定。在QIA表达系统中表达，Ni柱亲和层析纯化。进行生物学活性鉴定。结果: 经序列分析所获684 bp人canstatin基因与报道一致，重组进pQE30表达载体，IPTG诱导表达并纯化成功，表达产物能明显抑制血管生成。结论: 成功构建人canstatin cDNA克隆和表达载体并在大肠杆菌M15中高效表达。纯化回收产物在鸡胚绒毛尿囊膜（chorioallantoic membrane,CAM）实验中具有明显抑制血管生成活性

Cloning and Expression of Human Canstatin and Identification of Its Biological Activity

Objective:To clone and to express human canstatin gene and investigate biological activity of the recombi-nant protein. Methods:The cDNA of canstatin was amplified with RT-PCR from fresh fetal liver and was cloned into pMD18-T vector , and was sequenced. Then canstatin cDNA was cloned into the BamH I and Hind III sites of pQE30 and expressed with induction of IPTG. After the purification under native conditions, The biological activity of the recombinant protein was identified by the chick embryo chorioallantoic membrane assay (CAM). Results: The sequence of the amplified DNA fragment is consistent with that of the known gene. The recombinant protein was highly expressed after induction with IPTG. Biological assay results indicate that the recombinant canstatin protein could suppress the new blood vessel formation in CAM in vitro. Conclusion:The cloning and expression vector of canstatin cDNA has been constructed successfully. The recombinant canstatin protein was highly expressed in E. coli M15. In the CAM, the recombinant protein has highly suppressive effects on the vessels in chick embryo chorioallantoic membrane.