Plk1 PBD蛋白原核表达、分离纯化与活性鉴定

Bacterial expression,purification and biological activity evaluation of Plk1 PBD protein

目的:原核表达保罗样激酶1底物结合域(Polo-like kinase 1 Polo-box domain,Plk1 PBD)蛋白,经分离纯化后进行生物学活性鉴定。方法:以HeLa cDNA为模板经PCR反应扩增Plk1 PBD基因,利用DNA重组技术与pET-28a载体连接构建重组质粒。将重组质粒转化到E.coli Rosetta(DE3),经诱导培养后进行Plk1 PBD蛋白原核表达。采用亲和层析方法分离纯化Plk1PBD蛋白后,以酶联免疫吸附测定实验(enzyme linked immunosorbent assay,ELISA)和荧光偏振实验(fluorescence polarization,FP)进行生物学活性鉴定。结果:测序与重组质粒酶切结果表明,成功构建了pET-28a-Plk1 PBD原核表达质粒。SDS-PAGE电泳和Western blot实验证实了Plk1 PBD蛋白的原核表达。ELISA实验和FP实验表明Plk1 PBD蛋白具有良好的生物学活性。结论:成功进行了Plk1 PBD蛋白的原核表达、分离纯化与活性鉴定,为其生物学功能研究及以Plk1 PBD蛋白为靶标的新型抗肿瘤药物发现奠定了基础。

Objective :To develop the bacterial expression,purification and biological activity evaluation methods for Polo-like kinase 1 Polo-box domain(Plk1 PBD)protein.Methods:HeLa cDNA was served as a template to amplify Plk1 PBD gene fragment by PCR. After inserting into pET-28a vector,E.coli Rosetta(DE3)competent cells were transformed with pET-28a-Plk1 PBD plasmid.Plk1 PBD protein was expressed after induction and purified with HisTrap affinity chromatography,and then the biological activity was evaluated by enzyme linked immunosorbent assay(ELISA)and fluorescence polarization(FP)assay.Results:The DNA sequencing and double digestion map for recombinant plasmid showed the construction was successful.SDS-PAGE and Western blot assay demonstrated that Plk1 PBD protein was successfully expressed and purified,and ELISA and FP assay showed a perfect biological activity of Plk1 PBD protein.Conclusion:Plk1 PBD protein is prepared successfully,which paves the way for further study on its cellular functions in vitro and the development for novel Plk1 inhibitors targeting Polo-box domain.