Construction And Purification Of Tuberculosis Subunit Vaccine LT84

Object:We search for more effective vaccines to enhance the immunogenicity and protective efficacy of Mycobacterium bovis Bacille Calmette-Guerin (BCG). The five members of the Rpf-like family all have the resuscitation-promoting activity, all the five Rpf-like proteins of M. Tuberculos is the secreted protein. Resuscitation promoting factor D (Rpf D) is one of the five members of the Rpf-like family in M. Tuberculosis. According to the research, Rpf D is considered the most efficient one in the aspect of resuscitation-promoting activity. Therefore we selected Rpf D to enhance protective efficacy of tuberculosis subunit vaccine EAMMH. We clone and express recombinant protein ESAT6-Ag85B-RpfD-MPT64-Mtb8.4-HspX (EARMMH, LT84) in this experiment. Recombinant protein EARMMH(LT84) is purified for the object of control latent tuberculosis infection(LTBI). We hope this subunit vaccine will help people to control Mycobacterium tuberculosis (M. tuberculosis) in all stages of infection or even eradicate this disease.Method:The gene encoding protein RpfD (Rv2389c) was amplified from Plasmid pET30a-RpfD by PCR. The amplification product was purified and digested with Bgl II FastDigest, then inserted into plasmid vector pET30a-EAMMH to construct recombinant plasmid pET30a-EARMMH. The recombinant plasmid pET30a-EARMMH was transferred into E. coil DH5a strain for test and verified. After the test and verification, the correct plasmid was transferred into E. coil BL21strain. The purification of recombinant protein EARMMH(LT84) contain a series of steps such as collecting the broth, broken cells were centrifuged, resuspended bacterial.The purification of recombinant protein need GE AKTA Purifier UPC100and HiTraq HIC column.Results:We had construct the new recombinant plasmid pET30a-ESAT6-Ag85B-RpfD-MPT64-Mtb8.4-HspX successful. We express recombinant protein EARMMH(LT84) in E. coli BL21. The expression of fusion protein under the conditions like the induced temperature of34℃, the induction time4h, the DPTG final concentration of0.5mmol/L. The fusion protein EARMMH(LT84) was stably produced in E. coli BL21in supernatant. The purification of recombinant protein EARMMH(LT84) and was first purified by salting out and hydrophobic chromatography successively. After the purification we used the microplatereader measure the density of recombinant protein.The result of expression and purification of fusion protein EARMMH(LT84)had been identified by SDS-PAGE.Conclusion:The fusion protein EARMMH(LT84) was constructed, expressed and purified successfully. This experiment has made a new candidate vaccine for the development of M. tuberculosis subunit vaccine.