剪接因子1N端1-320氨基酸片段的原核表达及纯化

目的:通过扩增剪接因子1(SF1)的N端1-320氨基酸(aa)片段对应的cDNA,构建His融合蛋白原核表达质粒pET-28a(+)/SF1(1-320aa),在大肠杆菌中诱导表达并进行亲和纯化。方法:PCR扩增SF1的1-320 aa片段对应的cDNA,扩增产物和载体pET-28a(+)经酶切回收,连接载体和目的片段,获得重组质粒,转化大肠杆菌DH5α,挑取克隆、酶切鉴定、测序,将测序正确的重组质粒转化大肠杆菌BL21(DE3),IPTG诱导表达,SDS-PAGE和West-ern印迹分析蛋白表达情况,亲和纯化His-SF1(1-320aa)。结果:SF1片段以正确的读框插入pET-28a(+),IPTG可以诱导大肠杆菌表达重组蛋白,SDS-PAGE和Western印迹证实得到相对分子质量约为40×103的蛋白,亲和纯化得到高纯度蛋白质。结论:构建了His融合蛋白原核表达质粒pET-28a(+)/SF1(1-320aa),并获得His-SF1(1-320aa)融合蛋白,为进一步研究SF1和U2AF65之间的相互作用及对剪接体形成的影响提供了基础。

Prokaryotic Expression and Purification of N Terminal Amino Acids 1-320 Fragment of Splicing Factor 1

Objective： To construct prokaryotic expression vector,express and purify the His-tag and aa 1-320 of splicing factor 1（SF1） fusion protein in E.coli.Methods： The cDNA of SF1 aa 1-320 was amplified by PCR,and was inserted into pET-28a（＋）.After clone selection,the correct recombinant plasmid of pET-28a（＋） / SF1（1-320aa） was transformed into E.coli BL21（DE3） to express His-SF1（1-320aa）.The expressed protein was analyzed by SDS-PAGE and Western blotting,and was purified from the extract by affinity purification.Results： The SF1（1-320aa） cDNA was inserted into pET-28a（＋） with correct open read frame.The expression of His-SF1（1-320aa） could be induced by adding IPTG.The expressed protein showed a 43 kD major band by coomassie blue staining,which also was identified by anti-His or anti-SF1 antibody.Pure His-SF1（1-320aa） was obtained by affinity purification.Conclusion： His-SF1（1-320aa） is expressed and purified successfully from E.coli cells,which will help us to further study the interaction of SF1 and U2AF65,and the effect on the spliceosome formation.