Construction, Expression And Purification Of Tuberculosis Subunit Vaccine EAMMB

Objective To construct and express recombinant ESAT-6-Ag85B-MPT64(190-198)-MTB8.4-HBHA(31-i99)(EAMMB) in E.coil BL21(DE3) strain without affinity tag. EAMMB is purified for antigen bank of tuberculosis subunit vaccine.Method The gene encoding protein HBHA (containing SacI and HindⅢ) was amplified from BCG chromosomal DNA using PCR. The amplification product was purified and then inserted into plasmid vector pET30a-EAMM digested with SacI and HindⅢ to construct recombinant plasmid pET30a-EAMMB. The recombinant plasmid was transferred into E.coil DH5a strain for test and verify. Then the correct plasmid Import expression BL21strain.Take the best conditions of the expression to express protein, like the induced temperature of37°C, the induction time6h,the IPTG final concentration of0.1mmol/L. Using a series of steps such as collecting the broth, broken cells were centrifuged, resuspended bacterial, washed to obtain a certain purity of the target protein.Results The recombinant product EAMMB without any affinity tag apparent Molecular Weight is around of56kD, and it had been identified by SDS-PAGE.Conclusion The successful expression and purification of EAMMB has made a new candidate vaccine for the research of Mycobacterium tuberculosis subunit vaccine.