人纤溶酶原丝氨酸蛋白酶(SP)活性区基因的克隆、表达与功能研究  

Study on cloning,expression and function of recombinant human plasminogen serine protease activating region gene

作　　者：崔佳[1] 万翠香[2] 

机构地区：[1]长治医学院微生物学教研室,长治046000 [2]南昌大学食品科学与技术国家重点实验室

出　　处：《山西医科大学学报》2015年第10期982-985,共4页Journal of Shanxi Medical University

基　　金：江西省教育厅科研基金资助项目(GJJ13098)

摘　　要：目的克隆和表达人纤溶酶原(plasminogen,Plg)的丝氨酸蛋白酶(SP)活性区基因μplg,并进行重组蛋白的纯化及功能探索。方法用PCR方法从p DNR-plg质粒上扩增μplg,构建原核表达载体p ET22b(+)-μplg,IPTG诱导重组蛋白表达后复性,再经Ni+-NTA树脂亲和层析纯化,通过与双歧杆菌外膜蛋白Serpin的共孵育实验来探索μPlg的功能。结果 PCR成功扩增出了680 bp的人纤溶酶原Plg的SP区基因μplg,测序正确后与表达载体p ET22b(+)重组,重组质粒p ET22b(+)-μplg在大肠杆菌BL21中成功表达出分子量约为35 k D的μPlg蛋白质,经纯化后的蛋白纯度高达约90%以上,与Serpin蛋白共孵育后得到了二者的复合物。结论人纤溶酶原μPlg的成功克隆、表达及纯化对于研究SP区功能和与双歧杆菌的黏附作用有重要意义。Objective To clone,express and purify the serine protease activating region in human plasminogen（ Plg） encoded byμplg,and to explore the biological function of purified μPlg. Methods The gene encoding human plasminogen serine protease was amplified from p DNR-plg and the expression vector p ET22b（ ＋）-μplg was constructed by molecular biological method. Recombinant protein was expressed by IPTG induction and purified by Ni＋-NTA affinity chromatography. Finally,the biological function was explored after co-incubated with μPlg and Serpin. Results The 680 bp μplg was successfully cloned,and then reconstructed with the expression vector,naming p ET22b（ ＋）-μplg. The μPlg protein（ 35 k D） was successfully expressed in E. coli BL21. The purity of μPlg was up to 90% after affinity chromatography. After incubated with μPl and Serpin,the 70 k D complex was obtained. Conclusion The cloning,expression and purification of μPl may play an important role in understanding the mutual interaction / adhesion between human plasminogen SP activating region and bifdobacteria.

关 键 词：人纤溶酶原 丝氨酸蛋白酶(SP) 活性区 μplg 质粒构建 

分 类 号：Q78[生物学—分子生物学]