Prokaryotic expression,purification,and antigenic activity identification of Mycobacterium tuberculosis Rv2626c protein
Objective To express Mycobacterium tuberculosis Rv2626c protein in Escherichia coli(E.coli)and study the antigenicity of the purified recombinant Rv2626c protein.Methods The amino acid sequence of Rv2626c protein from Mycobacterium tuberculosis H37Rv strain(accession number:CCP45424.1)in GenBank was retrieved and converted into the corresponding DNA sequence according to the codon preference of E.coli.This DNA sequence was synthesized and cloned into pET24a(+)plasmid to construct pET24a(+)-Rv2626c recombinant plasmid.This plasmid was transformed into E.coli BL21(DE3)cells,and the expression of Rv2626c protein was induced under various conditions of isopropyl β-D-thiogalactopyranoside(IPTG)concentrations,temperature,and period.The recombinant Rv2626c protein was identified by SDS-PAGE and Western Blot.The recombinant Rv2626c protein was purified by nickel chelate affinity chromatography and used to immunize violet blue rabbits to prepare anti-Rv2626c anti-serum.The specificity and titer of the serum were respectively detected by Western Blot and enzyme-linked immunosorbent assay(ELISA).Results The recombinant plasmid pET24a(+)-Rv2626c was successfully constructed.SDS-PAGE analysis showed that recombinant Rv2626c was expressed in the recombinant plasmid transformed E.coli with IPTG induction,with a molecular weight of about 14 500,and the size was consistent with the expectation.The optimal expression condition for recombinant Rv2626c protein was at 31 ℃ with 1.0 mmol/L IPTG for 6 hours.The target protein was mainly present in a soluble form,which was consistent with the results of Western blot.The hyperimmunized serum with recombinant Rv2626c protein vaccination showed good specificity,with a titer of 1∶256 000 detected by ELISA.Conclusions Mycobacterium tuberculosis Rv2626c protein is successfully expressed in E.coli,and the purified protein has good purity and antigenic activity,laying the foundation for further reveals of its biological functions.
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