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

摘要:

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Plk1 PBD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。

Abstract:

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 e－valuated 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 demon－strated 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.