人S100A8和S100A9原核载体的构建、鉴定及蛋白纯化

Construction, identification and purification of recombinant prokaryotic expression vector expressing human S100A8 and S100A9

目的分别构建人S100A8(hS100A8)和S100A9(hS100A9)的PET-28a原核表达载体,并纯化hS100A8和hS100A9蛋白,为进一步研究hS100A8和hS100A9蛋白的分子生物学功能奠定实验基础。方法用带酶切位点的引物从hS100A8和hS100A9的真核表达载体扩增hS100A8和hS100A9基因片段,用相同的限制性内切酶双酶切PCR产物和pET-28a质粒。将两者用T4连接酶连接后转化入感受态细胞DH5α,提取质粒进行鉴定。将构建成功的pET28a-hS100A8和pET28a-hS100A9分别转化感受态细胞BL21,诱导蛋白表达,将等量纯化蛋白在一定钙离子浓度下室温孵育30 min,通过SDS-PAGE和Western blot验证异源二聚体是否形成。结果正确扩增出hS100A8和hS100A9基因,重组质粒pET28a-hS100A8和pET28a-hS100A9构建成功,纯化后在室温孵育后可形成异源二聚体。结论 hS100A8和hS100A9蛋白可以在体外形成异源二聚体,为探讨其生物学作用奠定理论基础。

Objective To construct recombinant prokaryotic expression vector expressing human S100A8（hS100A8） and human S100A9（hS100A9）, then purified recombinant hS100A8 and hS100A9 proteins, for further studying human its molecular biology effects. Methods Use the same restriction endonuclease double enzyme digestion PCR products and pET-28a plasmid, both connected with T4 ligase, then transformed into competent cell DH5α, extraction of plasmid for identification. The recombinant plasmid pET28 a-hS100A8 and pET28a-hS100A9 was confirmed by restriction endonuclease digestion, PCR and gene sequencing, then the resultant vectors respectively transformed into competent cell BL21. Induced the protein expression then isometric purified protein on calcium ion concentration must incubate for 30 min, by SDS-PAGE and Western blot methods validation of heterologous dimer formation. Results hS100A8 and hS100A9 gene was amplified accurately. Prokaryotic expression vector pET28a-hS100A8 and pET28a-hS100A9 were constructed successfully, and expressed in competent cell from Escherichia coli BL21, after purified at room temperature can be formed after incubation heterologous dimers. Conclusion The recombinant prokaryotic expression vectors expressing hS100A8 and hS100A9 was successfully constructed, and can form the heterologous dimers in vitro. These will be used to investigate the biological role of hS100A8 and hS100A9.