Cloning, Expressing Purifying Secreted Protein MPT64 Of Mycobacterim Tuberculosis.

Objective To clone the MPT64 gene of mycobacterium tuberculosis bovis,and construct prokaryotic plasmid of mycobacterium tuberculosis MPT64 and express it in E.coli,further more,to obtain high pure MPT64.Methods The whole MPT64 DNA sequence was amplified from mycobacterium tuberculosis bovis genome by polymerase chain reaction(PCR).Then the sequence was cloned into plasmid pET32a easy and sequenced.The recombinant expression plasmid pET32a-MPT64 was constructed and expressed in E.coli BL21,and MPT64 was purified by MagExtractor-His-tag Fusion Protein Purification Kit.Results The sequence of MPT64 was amplified and identical with that published in GenBank.The prokaryotic expresstion plasmid of pET32a-MPT64 had been constructed successfully.With induction of IPTG,a new fusion protein with relative molecular mass of 24000 was expressed and mainly located in cytoplasm,the expressed 6×His-MPT64 fusion proteins were identified by Western blotting with anti-His monoclonal antibody.Conclusions A confirmed MPT64 gene is cloned and expressed in E.coli successfully,and high pure MPT64 is obtained.This study provides a necessary condition for further research on MPT64 as antigen.