Fusion expression and purification of Mycobacterium tuberculosis difference protein MPT64 and ESAT6

    SHI Changª²Hong, WANG Xiaoª²Wu, ZHU Deª²Sheng, LI Yuan, XU Zhiª²Kai

    1Lab Animal Center, 2Department of Radiation Medicine, School of Preventive Medicine 3Department of Microbiology, School of Basic Medicine, Fourth Military Medical University, Xi¬ðan 710033, China

    ¡¾Abstract¡¿ AIM: To clone and fusion express secreted protein MPT64 and ESAT6 of Mycobacterium tuberculosis (MTB). METHODS: The gene encoding MPT64 and ESAT6 mature protein was amplified by polymerase chain reaction(PCR)from genome of Mycobacterium tuberculosis H37Rv strain, inserted into cloning vector pGEMª²Tª²easy for sequence analysis and then digested by restriction endonuclease and cloned into expression vector pProª²EX HT. The recombinant plasmid was transformed into expressive strain E.coli DH5¦Á and then induced with IPTG. The fusion protein was purified by Niª²NTA purification system. RESULTS: The sequences of mpt64 and esat6 amplified by PCR were identical with those reported in GenBank. The recombinant plasmid expressed fusion protein of MTB MPT64ª²ESAT6 had the relative molecular mass(Mr)of 38¡Á103, which was confirmed by Western blot analysis with speciesª²specific monoclonal antibody against 6¡ÁHis mAb. The fusion expressed protein was insoluble and could be purified by Niª²NTA purification system. CONCLUSION: Secreted protein MPT64ª²ESAT6 of Mycobacterium tuberculosis is successfully fusion expressed in E.coli DH5¦Á, which can be used for TB diagnosis. ......