山西医科大学学报
山西醫科大學學報
산서의과대학학보
Journal of Shanxi Medical University
2015年
10期
982-985
,共4页
人纤溶酶原%丝氨酸蛋白酶(SP)%活性区%μplg%质粒构建
人纖溶酶原%絲氨痠蛋白酶(SP)%活性區%μplg%質粒構建
인섬용매원%사안산단백매(SP)%활성구%μplg%질립구건
human plasminogen%serine protease%activating region%μplg%plasmid construction
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pDNR-plg质粒上扩增μplg,构建原核表达载体pET22b(+)-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pET22b(+)重组,重组质粒pET22b(+)-μplg在大肠杆菌BL21中成功表达出分子量约为35 kD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.
目的 剋隆和錶達人纖溶酶原( plasminogen,Plg)的絲氨痠蛋白酶( SP)活性區基因μplg,併進行重組蛋白的純化及功能探索. 方法 用PCR方法從pDNR-plg質粒上擴增μplg,構建原覈錶達載體pET22b(+)-μplg,IPTG誘導重組蛋白錶達後複性,再經Ni+-NTA樹脂親和層析純化,通過與雙歧桿菌外膜蛋白Serpin的共孵育實驗來探索μPlg的功能. 結果 PCR成功擴增齣瞭680 bp的人纖溶酶原Plg的SP區基因μplg,測序正確後與錶達載體pET22b(+)重組,重組質粒pET22b(+)-μplg在大腸桿菌BL21中成功錶達齣分子量約為35 kD的μPlg蛋白質,經純化後的蛋白純度高達約90%以上,與Serpin蛋白共孵育後得到瞭二者的複閤物. 結論 人纖溶酶原μPlg的成功剋隆、錶達及純化對于研究SP區功能和與雙歧桿菌的黏附作用有重要意義.
목적 극륭화표체인섬용매원( plasminogen,Plg)적사안산단백매( SP)활성구기인μplg,병진행중조단백적순화급공능탐색. 방법 용PCR방법종pDNR-plg질립상확증μplg,구건원핵표체재체pET22b(+)-μplg,IPTG유도중조단백표체후복성,재경Ni+-NTA수지친화층석순화,통과여쌍기간균외막단백Serpin적공부육실험래탐색μPlg적공능. 결과 PCR성공확증출료680 bp적인섬용매원Plg적SP구기인μplg,측서정학후여표체재체pET22b(+)중조,중조질립pET22b(+)-μplg재대장간균BL21중성공표체출분자량약위35 kD적μPlg단백질,경순화후적단백순도고체약90%이상,여Serpin단백공부육후득도료이자적복합물. 결론 인섬용매원μPlg적성공극륭、표체급순화대우연구SP구공능화여쌍기간균적점부작용유중요의의.
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 pDNR-plg and the expression vector pET22b(+)-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 ex-plored after co-incubated with μPlg and Serpin. Results The 680 bp μplg was successfully cloned, and then reconstructed with the expression vector, naming pET22b(+)-μplg. TheμPlg protein(35 kD) was successfully expressed in E. coli BL21. The purity ofμPlg was up to 90% after affinity chromatography. After incubated withμPl and Serpin, the 70 kD 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.
