【摘要】 目的:在大肠杆菌中表达人源转录因子sp1蛋白,并进行体外纯化。方法:首先将人源sp1真核表达质粒pCMV-sp1中的sp1 cDNA用限制性内切酶切开,连入原核表达质粒pET28b,构建原核表达重组质粒pET28b-sp1-699c;然后将重组质粒转化入大肠杆菌BL21(DE3)菌株,经IPTG诱导表达带His-Tag的融合蛋白His-sp1(88-786aa),通过包涵体的变复性和Ni-IDA亲和层析纯化后,SDS-PAGE鉴定纯化后的蛋白。结果:融合蛋白His-sp1在大肠杆菌内得到高效表达,纯化后可得到较高纯度的蛋白。结论:本研究获得了较高纯度的融合蛋白His-sp1,为进一步研究sp1蛋白对靶基因的转录调控奠定了基础。更多还原

【Abstract】 Objective: To express human transcription factor sp1 in E. coli and purify this protein. Methods:( 1) Prokaryotic expression recombinant plasmid pET28b-sp1-699c was constructed,while cutting the cDNA of sp1 from human sp1 eukaryotic expression plasmid pCMV-sp1 with restriction enzymes and inserting into prokaryotic expression plasmid pET28b by T 4 DNA ligase.( 2) This recombinant plasmid was transform into BL21( DE3) E. coli strain,expression of His-Tag fusion protein His-sp1 with IPTG induction.( 3) The products by denaturation and renaturation of inclusion body,and affinity chromatography were purified,and then the purified protein was detected by SDS-PAGE. Results: Fusion protein His-sp1 was expressed efficiently in E. coli,and protein with high purity was obtained. Conclusion: In this study,we obtained fusion protein His-sp1 with high purity and quantity, laid a foundation for further research of the transcription regulation sp1 target gene.更多还原