人骨保护素-分枝杆菌热休克蛋白70融合蛋白的克隆与表达及其活性鉴定  

Cloning, expression and activity identification of human osteoprotegerin-mycobacteria heat shock protein 70 fusion protein

作　　者：马静[1] 王炜[1] 赵文明[1] 李慎涛[1] 曾牧[1] 刘振龙[1] 

机构地区：[1]首都医科大学免疫学系,北京市100069

出　　处：《中国组织工程研究与临床康复》2008年第15期2903-2909,共7页Journal of Clinical Rehabilitative Tissue Engineering Research

基　　金：北京市优秀人才培养资助项止(20061D0501800258);北京市教委科技发展计划项目资助(KM200810025013)~~

摘　　要：目的:为解决类风湿性关节炎中骨破坏及炎症损伤两大难题,拟克隆人骨保护素功能区与分枝杆菌热休克蛋白70肽段融合基因,进一步观察其在大肠杆菌中的表达并鉴定活性。方法:实验于2006-05/2007-09在首都医科大学免疫学系实验室完成。采用反转录-聚合酶链反应技术从人骨肉瘤细胞系MG63总RNA中扩增骨保护素成熟肽段编码区基因,构建pGEM-TEasy-骨保护素重组质粒。以此为模板,聚合酶链反应扩增骨保护素-热休克蛋白70功能区DNA,构建重组表达载体pET-28a-骨保护素-热休克蛋白70,将其转化E.coli.BL21(DE3),经异丙基硫代半乳糖苷诱导后收集菌体蛋白,以十二烷基硫酸钠-聚丙烯酰胺凝胶电泳及蛋白免疫印迹鉴定该融合蛋白的表达,以破骨细胞生长抑制实验及抑炎实验检测该融合蛋白的生物学活性。结果:①实验最终获得人骨保护素全长基因片段,人骨保护素-热休克蛋白70功能区DNA片段已被正确插入到pET-28a载体中,转化菌株可表达人骨保护素-热休克蛋白70融合蛋白。②十二烷基硫酸钠-聚丙烯酰胺凝胶电泳分析显示约在Mr22000处有蛋白的超表达,而未诱导菌株未发现有此条带。③蛋白免疫印迹检测表明,融合蛋白能与抗人骨保护素单克隆抗体特异性结合。④破骨细胞生长抑制实验表明,该融合蛋白能够减少破骨细胞的生成数量,具有体外抑制骨破坏的生物活性。⑤抑炎实验表明,融合蛋白具有显著减轻迟发型超敏反应小鼠模型炎症反应程度的作用,说明融合蛋白中热休克蛋白70具有抑制炎症的生物学活性。结论:在E.coli.BL21(DE3)中表达了骨保护素-热休克蛋白70融合蛋白,体外功能实验证实该融合蛋白具有一定的生物学活性。AIM： To solve the two difficulties of bone resorption and inflammation in rheumatoid arthritis, clone the recombinant human osteoprotegerin （OPG） and mycobacteria heat shock protein 70 （HSP70） functional gene, and study the expression and activity of OPG-HSP70 fusion protein in E.coli. METHODS： Experiments were performed at the Laboratory of Department of Immunology, Capital Medical University from May 2006 to September 2007. Complementary DNA encoding full length OPG protein was amplified by reverse transcription-polymerase chain reaction （RT-PCR） from human osteosarcoma cell line MG63 and cloned into pGEMT-Easy vectors. Then, using the recombinant plasmid as the template, the DNA encoding the fusion protein OPG-HSP 70 was amplified by PCR, and was inserted into prokaryotic expression vector pET-28a. Construct pET-28a-OPG-HSP 70 was used to transform into competent E.coli. BL21（DE3） which were induced by isopropyl B-D-thiogalactopyranoside （IPTG）, and the fusion protein from above E.coli was collected. Sodium dodecyl sulfate polyacrylamide gel electropheresis （SDS-PAGE） and Western-blotting were performed to identify OPG-HSP 70 fusion protein. The experiments of osteoclast inhibition and restraining inflammation were used to detect the bioactivity of fusion protein. RESULTS： （1）The complementary DNA encoding full length OPG protein was obtained. OPG-HSP 70 fusion gene obtained in this experiment was successfully inserted into pET-28a vector. OPG-HSP70 fusion protein was expressed when transformed into E.coli.BL21（DE3）. （2）SDS-PAGE indicated that the fusion protein was large expressed at the molecular weight of Mr22 000, but there were no band in the total lysate of bacteria harboring pET-28a-OPG-HSP70 without IPTG induction group. （3）Western-blotting indicated that the OPG-HSP70 fusion protein could specifically react with anti-human OPG monoclonal antibodies. （4）The osteoclast inhibition test demonstrated that the fusion protein could reduce the number of osteoc

关 键 词：在E.coli.BL21(DE3)中表达了骨保护素-热休克蛋白70融合蛋白 体外功能实验证实该融合蛋白具有一定的生物学活性. 

分 类 号：R593.22[医药卫生—内科学]