Prokaryotic expression, purification and immunoreactivity of Mycobacterium tuberculosis Ag85A protein / 基础医学与临床

ABSTRACT

Objective To obtain M. Tuberculosis Ag85A protein by prokaryotic expression. Methods The fbpA gene encoding M. Tuberculosis Ag85A protein was amplified by polymerase chain reaction ( PCR) from M. Tuberculosis H37R_V strain. The PCR product was cloned into prokaryotic expression vector pProEXHTb to generate the recombi-nant plasmid pProfbpA, which was then transformed into the competence Escherichia coli BL21 cells. The recombi-nant Ag85A protein was successfully expressed by isopropyl thio-β-D-galactoside(IPTG) induction and purified by the Ni-purification system. The distribution of fbpA gene in different nonpathogenic mycobacterial strains was screened by PCR and ELISA was performed to determine the immunoreactivity of the recombinant Ag85A protein with serum from mice with mycobacterial infections. Results 32 ku Ag85A protein was successfully expressed and purified. It was confirmed by PCR and ELISA that fbpA gene presented in the genomes of M. Tuberculosis H37Rv, H37Ra, BCG, M. Smegmatis, M. Terra, M. Trivial and M. Phlei, but being absent in the genomes of M. Vaccae. The highest Ag85 A antibody titer was found in serum of TB patients and mice infected by M . Tuberculosis H37Rv .Conclusion The recombinant Ag85A protein was successfully expressed and purified.