~1PEP-1-p27mt融合蛋白的原核表达及纯化

目的:构建融合蛋白PEP-1-p27mt的原核表达质粒,并进行诱导表达和蛋白纯化。方法:通过PCR方法自pORF9-hp27mtv21质粒中扩增p27mt基因,克隆入中间载体pGEM-TEasy Vector。经XhoI和BamH1双酶切后,构建原核表达载体pET15b-pep-1-p27mt,经测序证实构建成功后转化EcoliBL21(DE3)pLysS,表达PEP-1-p27mt融合蛋白,经Ni-NTA树脂亲合层析,纯化产物进行SDS-PAGE及Western Blot分析鉴定。结果:成功构建PEP-1-p27mt融合蛋白原核表达载体,表达并纯化了相对分子量为32×103的PEP-1-p27mt融合蛋白。结论:PEP-1-p27mt融合蛋白表达载体的构建及目的蛋白的表达纯化,为体内应用细胞周期抑制蛋白诱导肿瘤细胞凋亡的研究提供了理论基础。

Prokaryotic expression and purification of PEP-1-p27mt fusion protein

Objective:To construct prokaryotic expression vector pET15b-pep-1-p27mt and to purify the fusion protein of PEP-1-p27mt.Methods: To amplify p27mt cDNA from the plasmid of pORF 9-hp27mt by PCR .Then the amplified p27mt cDNA was ligated with pGEM-T easy vector . The pET15b-pep-1 and pGEM-T Easy-p27mt were excised with BamH I and Xho I to construct pET15b-pep-1-p27mt.The constructed recombinant plasmids were identified by sequencing and transformed into E coli BL21(DE3)pLysS for expression of PEP-1-p27mt fusion protein.After purified by Ni2+-resin affinity chromatography,expressed products were identified by SDS-PAGE and Western blot.Results: The recombinant plasmids were constructed and PEP-1-p27mt fusion protein with relative molecular weights of 32×103 was expressed successfully.Conclusion: Construction of recombinat plasmid pET15b-pep-1-p27mt and expression and purification of the PEP-1-p27mt fusion protein provide a basis for using cyclin dependent kinase inhibitors p27mt in the inducing apoptosis of tumor cells.