Construction,prokaryotic expression,purification and bioinformatic predication of the recombinant plasmid carrying phoS2 gene from Mycobacterium tuberculosis / 西安交通大学学报(医学版)

ABSTRACT

Objective To construct a recombinant plasmid carrying phoS2 from Mycobacterium tuberculosis, to express prokaryotically,to purify the recombinant protein and to identify the immunogenicity of its recombinant protein.Methods phoS2 gene was subcloned into expression vector pET32a.The constructed recombinant plasmid phoS2/pET32a was transformed to E.coli BL21 (DE3)for expression under induction of IPTG.The expressed product was identified by SDS-PAGE and then purified by His-bind affinity chromatography with Ni2+.Results of the induced expression level were detected by SDS-PAGE. The recombinant protein was purified by affinity chromatography.The biological activity of phoS2 was tested by Western blot;its secondary structure and 3-D structure were predicted with software packages such as DNAstar and Rasmol.Results The recombinant plasmid phoS2/pET32a of Mycobacterium tuberculosis was constructed successfully and confirmed by restriction endonuclease analysis and DNA sequencing analysis. SDS-PAGE analysis showed that the expressed proteins were mainly insoluble;Western blot analysis showed that the molecular weight of phoS2 recombinants protein was 3 7 9 5 3 u.The recombinant protein was purified by affinity chromatography.Analysis of the predicted protein indicated that the molecular mass was 37 953.1 ku,PI was 5.75,there was one transmembrane region and function sites were 1 to 370. Conclusion The recombinant plasmid phoS2/pET32a of Mycobacterium tuberculosis was successfully constructed and phoS2 gene could be expressed in BL2 1 with high efficiency.Predicting protein structure and function can provide some theoretical basis for conducting the present experiment and selecting further research.