Prokaryotic expression, purification and identification of human procalcitonin
Objective To construct the prokaryotic expression vector of human procalcitonin(PCT) and obtain the highly purified recombinant PCT protein.Methods PCT primers were designed based on the sequence of PCT gene from NCBI,and PCT gene was amplified by PCR.Then,the recombinant vector PCT/pET-22b(+) was constructed and transferred into E.coli BL21 to induce the expression of recombinant PCT protein.Last,the recombinant PCT protein was purified by the nickel column affinity chromatography and identified by Western blot and the colloidal gold method,respectively.Results The results of agarose gel electrophoresis showed that the product of PCR amplification was about 350 bp.The homology comparison analysis revealed that the PCT gene fragment(348 bp) was successfully inserted into pTG19-T vector,and that no any base mutation was found.When the recombinant plasmid of PCT/pET-22b(+) was digested with BamH Ⅰ and HindⅢ,two pieces of about 350 bp and 5 500 bp were obtained.SDS-PAGE showed that the recombinant PCT protein with about 14 000 of Mr was existed in soluble form,and was easily obtained by the nickel column affinity chromatography.Moreover,western blot and the colloidal gold method demonstrated that the recombinant PCT protein was successfully expressed and contained histidine label(His-tag).Conclusion The recombinant vector PCT/pET-22b(+) is successfully constructed by the recombinant DNA technology,and the recombinant PCT fusion protein is successfully obtained by the nickel column affinity chromatography.
NETL
NSTL
万方数据
维普
