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Chinese Journal of Biologicals. 2011, (09)

Prokaryotic Expression and Purification of MPT64 Protein of Mycobacterium bovisChinese Full Text

TAN Wei,NIE Ying,WU Ren-biao,CHENG Li-qing,YAN Guang-mou,ZHANG Xi-chen,ZHANG Rui(College of Animal Science and Veterinary Medicine,Jilin University,Changchun 130062,China)

Abstract: Objective To express the MPT64 protein of Mycobacterium bovis in prokaryotic cells and purify the expressed product.Methods MPT64 gene was amplified by PCR using the whole genomic DNA of M.bovis Vallee strain as a template and cloned into vector pET-28a（+）.The constructed recombinant plasmid pET-MPT64 was transformed to E.coli BL21（DE3） for expression under induction of IPTG.The expressed product was identified by SDS-PAGE,then purified by Ni-NTA affinity chromatography and further identified by Western blot.Results Sequencing result showed the homology of nucleotide of obtained MPT64 gene to that reported in GenBank was 100%.Restriction analysis proved that recombinant plasmid pET-MPT64 was constructed correctly.The expressed recombinant MPT64 protein,with a relative molecular mass of about 26 000,mainly existed in a soluble form and contained about 11% of total somatic protein.Purified recombinant MPT64 protein reached a purity of 93% and showed specific reaction with bovine serum against M.bovis.Conclusion The MPT64 protein of M.bovis was successfully expressed in prokaryotic cells and purified,which might be used as an antigen for diagnosis of novel bovine tuberculosis.
Keywords：

Mycobacterium bovis; MPT64 protein; Prokaryotic cells; Gene expression; Purification;

  • DOI：

    10.13200/j.cjb.2011.09.63.tanw.016

  • Series：

  • Subject：

  • Classification Code：

    R392.1

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