人内皮抑素基因改造、表达、纯化及活性检测

目的克隆诱变的人内皮抑素(human endostatin,hES)氨基端基因,并检测其活性。方法双酶切已诱变的人内皮抑素基因,电泳回收氨基端片段,与质粒pTYB-2重组,转化E．coli BL-21(DE3)。通过鸡胚绒毛尿囊膜(CAM)实验,MTT实验,HE染色及流式细胞术检测重组小分子人内皮抑素对新生血管生成、细胞增殖和细胞凋亡的影响。结果基因重组小分子内皮抑素对鸡胚尿囊膜新生血管生成具有明显的抑制作用；对脐静脉内皮细胞和肝癌细胞增殖的抑制存在量效关系；作用24h后,观察到细胞凋亡现象；与对照组相比,细胞凋亡数增加。结论成功构建了人内皮抑素氨基端基因工程菌,得到了具有生物活性的氨基端小分子内皮抑素,为便利临床应用奠定了基础。

Gene Modification, Expression and Purification of Human Endostatin and Its Biological Activity

Objective To clone the N-terminal sequence of mutant-type human endostatin and investigate its biological activities.Methods The mutant-type human endostatin gene was digested by two restriction endonucleases and the N-terminal gene fragment was recovered by electrophoresis.Then the fragment was cloned into the E.coli expressing plasmid pTYB-2, transformed into E.coli strain BL21 (DE3) and expressed by IPTG induction.After purification by one-step affinity chro-matography, the small endostatin protein was obtained and its activity was analyzed by chick embryo chorioallantoic membrane (CAM).The effect of the small endostatin on cell lines was examined by using MTT assay.The morphological change of cells was observed by HE staining.The apoptosis of cells was detected by the flow cytometry.Results The angiogenesis of CAM was inhibited by the small endostatin protein.MTT assay showed that the inhibition was obviously enhanced as the concentration increased.Morphological observation showed that apoptosis happened after the cells were treated with the protein for 24 hours and the small endostatin increased the apoptotic cell.Conclusion The recombinant N-terminal sequence of mutant-type human endostatin was transformed into the host stain E.Coli and the N-terminal small protein has biological activity.