Tumstatin_(183-230)-TRAIL融合蛋白的克隆表达及其生物学双功能鉴定  

Expression of Tumstatin_(183-230)-TRAIL fusion protein and identification of its biological functions

作　　者：任娜[1] 王梁华[1] 高云[1] 孙铭娟[1] 焦豫良[1] 郭爱云[1] 焦炳华[1] 

机构地区：[1]第二军医大学基础部生物化学与分子生物学教研室,上海200433

出　　处：《第二军医大学学报》2008年第5期474-478,共5页Academic Journal of Second Military Medical University

基　　金：上海市科学技术发展基金(024319115)~~

摘　　要：目的:克隆表达融合蛋白Tumstatin_(183-230)-TRAIL,并观察其生物学功能。方法:利用重组PCR技术扩增Tumsta- tin_(183-230)-TRAIL(Tu-T)的融合编码序列.构建pMAL—Tu—T融合蛋白表达载体,将其转化大肠杆菌BL21(DE3)。经IPTG诱导表达,得到MBP—Tu—T融合蛋白.用SDS-PAGE鉴定纯化的表达产物。通过体外内皮细胞增殖抑制实验、肿瘤细胞杀伤实验、体外管腔形成抑制试验及电镜检测细胞凋亡情况对其生物学双功能进行鉴定。结果:融合蛋白MBP—Tu—T在BL21中表达率约20%.可明显抑制内皮细胞增殖(IC_(50)12.5μg/ml),杀伤、诱导胰腺癌细胞凋亡并抑制体外管腔形成。结论:融合蛋白MBP-Tu-T具有双功能生物学活性,为进一步研究其靶向性杀伤肿瘤奠定了基础。Objective：To express Tumstatin183 230-TRAIL fusion protein and to observe its biological functions. Methods： SOE-ing PCR was employed to amplify the recombinant sequence of Tumstatin183-230 and TNF-related apoptosis-inducing ligand （TRAIL114-281）. An expression vector pMAL-Tu-T was constructed by inserting Tu-T sequence into pMAL-c2 ; the vector was used to transfect E. coli BL21 （DE3） and expression of MBP-Tu-T fusion protein was induced by IPTG. Amylose Resin columns were employed to purify the fusion protein. The biological functions of MBP-Tu-T protein was examined by inhibitory test of endothelial cell proliferation, standard tumor cell cytotoxic assay, in vitro tube formation inhibition, and electron microscopic observation （apoptosis）. Results： The expression rate of MBP-Tu-T fusion protein in E. coli was about 20%. Purified recombinant protein obviously inhibited endothelial cell proliferation （IC50 12. 5 μg/ml ）, induced apoptosis of pancreatic cancer cells, and inhibited tube formation. Conclusion.. Constructed MBP-Tu-T fusion protein is bifunctional, which lays a solid foundation for further investigation of antitumor effect of Tumstatin183 230-TRAIL in vivo.

关 键 词：Tumstatin183-230-TRAIL 融合蛋白 生物学功能 

分 类 号：Q784[生物学—分子生物学]