瑞替普酶融合蛋白的表达、纯化及分子伴侣对其复性的影响  

Reteplase fusion protein: expression,purification and the effect of chaperones on its renaturation

作　　者：王伟[1] 邹文艺[2] 范清林[2] 宋礼华[1,2] 

机构地区：[1]安徽医科大学生物化学与分子生物学教研室,合肥230032 [2]安徽安科生物工程(集团)股份有限公司,合肥230088

出　　处：《安徽医科大学学报》2013年第11期1287-1291,共5页Acta Universitatis Medicinalis Anhui

摘　　要：目的构建瑞替普酶(r-PA)融合蛋白的原核表达体系,并研究DsbA、DsbC、pKJE7、pGro7、pTf16等分子伴侣对其复性的影响。方法将r-PA基因克隆到表达载体质粒PET-32a中,通过异丙基硫代半乳糖苷(IPTG)诱导,在宿主菌E.coli BL21中进行表达。在复性时分别添加不同分子伴侣,研究不同分子伴侣对融合蛋白体外复性的影响。结果SDS-PAGE和Western blot结果表明r-PA融合蛋白正确表达,分子伴侣DsbA、pKJE7、pTf16对融合蛋白的复性有明显效果,体外溶圈实验表明复性后的r-PA具有纤溶活性。结论分子伴侣能有效帮助r-PA融合蛋白的复性。Objective To construct the prokaryotic expression system of reteplase fusion protein and research the effect of chaperones on its renaturation. Methods Inserted the reteplase gene into the prokaryotie expression vector PET32a and then expressed it by the induction of IPTG in E. coli B1221. Researched the effect of chaperones on the renaturation of fusion protein by adding different chaperones. Results The analysis of SDS-PAGE and Western blot indicated that reteplase fusion protein was expressed correctly. Chaperones DsbA ,pKJE7 ,pTf16 had the conspicuous effect on the renaturation of fusion protein. The result of activity assay indicated that the refolded reteplase fusion protein had fibrinolytic activity. Conclusion Chaperones can promote renaturation of reteplase fusion protein.

关 键 词：瑞替普酶 融合蛋白 分子伴侣 复性 

分 类 号：Q789[生物学—分子生物学]