人Kin17蛋白的原核表达及纯化

Prokaryotic Expression and Purification of Human Kin17 Protein

目的:构建重组质粒,在原核系统中表达带His标签的人Kin17融合蛋白,并对表达产物进行纯化及鉴定。方法:以PK3质粒为模板,PCR扩增出全长的KIN17基因,并将PCR产物插入到原核表达载体pET32a中,构建的重组质粒经BamHⅠ/HindⅢ双酶切及DNA测序方法鉴定正确后,转化至大肠杆菌BL21,并用丙基-β-硫代半乳糖苷诱导表达His-Kin17融合蛋白,最后应用Ni-NTA柱亲和层析方法来纯化融合蛋白及经Western免疫印迹鉴定纯化的重组蛋白。结果:双酶切及DNA测序方法证实pET32a-KIN17质粒构建正确,融合蛋白经诱导表达、纯化、鉴定后,得到纯度达到95%以上的His-Kin17重组蛋白。结论:成功构建携带KIN17基因的重组载体,并获得了高纯度的His-Kin17重组蛋白,为深入研究Kin17蛋白的分子结构、功能及其在肿瘤发生发展中的作用奠定了基础。

Objective： To express and purify the human Kinl7 fusion protein with His label by constructing recombinant plasmid in E. coll. Method：AU length of K/N17 gene was amplified from PK3 plasmid by PCR. And the PCR product was cloned into Prokaryotic vec- tor pET32a. After determined by double digestion and DNA sequencing, the recombinant plasmid pET32a - KIN17 was transformed into the prokaryotic expression cells BL21. Kinl7 protein induced with isopropy - β- D -thiogalaetoside （IPTG） in E. coli was purified on Ni -NTA resin column and determined by SDS- PAGE method and Western blot assay. Result：The recombinant plasmid pET32a- KIN17 carrying K/N17 gene was successfully constructed according to double digestion and DNA sequencing. After induction, purification and i- dentification, the purity of Kin17 fusion protein acquired was above 95 %. Conclusion：The recombinant plasmid pET32a - KIN17 carrying human K/N17 gene is constructed successfuUy, and the Kin17 recombinant protein with high purity was obtained in E. coli. This work will lay the foundation for the deep - going study of the molecular structure, function of Kin17 protein and its roles in the cancer development.