CAJ | 학술논문

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。
목적구건서체보매(r-PA)융합단백적원핵표체체계，병연구DsbA、DsbC、pKJE7、pGro7、pTf16등분자반려대기복성적영향。방법장r-PA기인극륭도표체재체질립PET-32a중，통과이병기류대반유당감( IPTG)유도，재숙주균E. coli BL21중진행표체。재복성시분별첨가불동분자반려，연구불동분자반려대융합단백체외복성적영향。결과 SDS-PAGE화Western blot 결과표명 r-PA 융합단백정학표체，분자반려DsbA、pKJE7、pTf16대융합단백적복성유명현효과，체외용권실험표명복성후적r-PA구유섬용활성。결론분자반려능유효방조r-PA융합단백적복성。
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 BL21. 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 conspicu-ous effect on the renaturation of fusion protein. The result of activity assay indicated that the refolded reteplase fu-sion protein had fibrinolytic activity. Conclusion Chaperones can promote renaturation of reteplase fusion protein.