人膀胱癌组织Kringle 5基因的克隆、优化表达、纯化及活性鉴定  

Cloning,Optimal Expression,Purification and Activity Identification of Plasminogen Kringle 5 from Human Bladder Carcinoma Tissue

作　　者：柯昌兴[1,2] 王剑松[1,2] 胡明宇[1,2] 毕城伟[1,2] 陈慧诚[3] 刘龙丁[3] 颜汝平[1,2] 詹辉[1,2] 

机构地区：[1]昆明医学院第二附属医院泌尿外科 [2]云南省泌尿外科研究所,云南省昆明市650101 [3]中国医学科学院医学生物学研究所病毒免疫室

出　　处：《中国全科医学》2010年第15期1656-1659,共4页Chinese General Practice

基　　金：云南省卫生科技计划项目(2009NS068);昆明医学院博士创新基金资助(KM2008D04)

摘　　要：目的构建能够表达分泌性人纤溶酶原krinle5(K5)的大肠杆菌工程菌,优化表达并鉴定其抗人血管内皮细胞(ECV)304的生物活性。方法从人膀胱癌组织中提取总RNA,RT-PCR扩增K5基因,构建原核表达载体pGEX-5X-1/K5,运用工程菌表达蛋白。带谷胱甘肽(GST)标签琼脂糖4B亲和柱层析、纯化蛋白,CellTiter96AQueous细胞增殖检测法(MTS)检测K5对ECV304增殖影响。结果 PCR扩增得到282bp片段,并成功插入pGEX-5X-1质粒,在异丙基-β-D-硫代半乳糖苷(IPTG)诱导下工程菌表达了特异性融合蛋白,经凝血酶酶切后得到分子量约为12kD的K5蛋白;K5蛋白能特异性抑制ECV304增殖,抑制率最高蛋白浓度为5ng/L。结论含重组质粒pGEX-5X-1/K5大肠杆菌表达产物量多且易纯化,为包括肿瘤在内的病理性血管增生疾病抗血管生成药物开发做了积极的尝试。Objective To establish an engineering E-coli strain which could produce secretary human plasminogen kringle 5 （K5）,to optimize expression parameters and identify the anti-proliferative effects of K5 on endothelial cell of vessels（ECV304）.Methods Total RNA was extracted from human bladder carcinoma tissue,then the K5 cDNA was amplified by RT-PCR.A prokaryotic expression vector pGEX-5X-1/K5 was constructed and the K5 protein was expressed in engineering bacteria.K5 was purified by tagged GST agarose 4B affinity column chromatography,the anti-proliferative effects of K5 on ECV304 was examined by Cell Titer 96？ Aqueous Non-Radioative Cell Proliferation Assay （MTS）.Results The acquired gene was 282 bp and it was inserted into pGEX-5X-1 plasmid successfully.The E-coli containing recombinant vector expressed fusion protein after inducted by IPTG,K5 protein,molecular weight of 12 kD was obtained after digested by thrombin.K5 protein showed effective in specifically inhibiting proliferation of ECV304 and its optimum concentration was 5μg/ml.Conclusion K5 protein has a large expression level and easy to purify by way of E-coli containing recombinant plasmid pGEX-5X-1/K5.It made a positive attempt to exploit anti-angiogenesis drugs to treat pathological angiogenesis diseases including cancers.

关 键 词：纤溶酶原 Kringle5基因 融合蛋白 基因 

分 类 号：R730.5[医药卫生—肿瘤] R737.14[医药卫生—临床医学]