NK4蛋白在大肠埃希菌中的表达、纯化、复性及活性测定

Expression,purification,renaturation and activity assay of NK4 in Escherichia coli

目的在大肠埃希菌(E.coli)中高表达NK4蛋白,经鉴定、纯化、复性后测定其生物学活性。方法采用重组DNA技术对已有质粒pGEX-4T-1-NK4和pBV220-HGFα进行改造,构建质粒pBV220-NK4,并使其转化E.coliBL21(DE3),温控诱导表达目的蛋白NK4。蛋白鉴定采用SDS-PAGE和Western blot;蛋白纯化采用包涵体超声破碎、洗涤和Sephacryl-S200凝胶过滤层析。经梯度稀释复性后,采用MTT法和免疫细胞化学方法检测重组蛋白NK4对人脐静脉内皮细胞(human umbilical vein endothelial cell,HUVEC)ECV304增殖的作用。结果重组质粒pBV220-NK4酶切图谱和序列测定与预期一致。SDS-PAGE显示有相对分子质量49 000的目的蛋白,表达量为30%。Western blot结果证实目的蛋白带有NK4的氨基端,经包涵体洗涤、层析后NK4的纯度为95%。复性后的NK4可抑制ECV304增殖,并抑制ECV304增殖细胞核抗原(proliferating cell nuclearantigen,PCNA)的表达。结论我们建立了NK4的原核高表达体系及纯化制备工艺,所制备的NK4蛋白具有生物学活性,为大规模制备有活性的NK4及深入研究其相关功能奠定了基础。

Objective To construct a recombinant plasmid expressing NK4 gene in Escherichia coli（E.coli） and to obtain purified NK4 protein with bioactivity. Methods The plasmid pBV220-NK4 was constructed by recombinant technology with utilization of the plasmid pBV220-HGFα and pGEX-4T-1-NK4.The plasmid pBV220-NK4 was identified by the restriction analysis and DNA sequencing.Then recombinant plasmid was transformed into E.coli and the recombinant protein was expressed and detected by SDS-PAGE and Western blot.The expressed protein existing in the form of inclusion body was purified by gel filtration chromatography,and then was detected for the activity by MTT method after renaturation.The expression of the proliferating cell nuclear antigen（PCNA） in human umbilical vein endothelial cells（HUVEC） ECV304 incubated with NK4 was measured by immunohistochemical strain.Results SDS-PAGE showed a specific protein band with relative molecular weight of 49 000.The expressed product existed in the form of insoluble inclusion body and contained about 30% of total somatic protein.After renaturation,the expressed NK4 showed an inhibitory action on ECV304 proliferation by MTT and the ratio of inhibition was 30%（P0.01）.The expressed NK4 also obviously depressed the expression of PCNA. Conclusions NK4 protein was successfully expresseed in E.coli,which layed the foundation for the study on the structure and function as well as pilot production of NK4.