[High-level expression of Mycobacterium tuberculosis 16,000 antigen in E. coli]
1Tuberculosis Research Laboratory, 309th Hospital of PLA, Beijing 100091.
Objective:
To gain the recombinant protein antigen 16,000 of Mycobacterium tuberculosis highly expressed in E. coli and study its immunological characteristics.
Methods:
DNA fragments code for the protein were obtained by PCR, then cloned into the pET plasmid vector to gain recombinant E. coli. Cells were cultured and induced to produce recombinant protein, whose molecular size and present form were analyzed by SDS-PAGE, and its immunological characteristics were analyzed by Western-blotting and ELISA technology.
Results:
The clone was analyzed at the nucleotide lever and shown the same DNA sequence coding for natural 16,000 protein. Analyzed by SDS-PAGE and Western-blotting, it was found to produce immunoreactive proteins with mobilities very similar to those of the 16,000 protein antigen, and the recombinant protein amounted to 40% of total cell proteins. ELISA results indicated that the purified recombinant protein could distinguish sera from tuberculosis patients with anti-PPD antibody and those from tuberculin positive contacts.
Conclusions:
Recombinant 16,000 protein antigen highly expressed in the form of solution in E. coli was gained and this antigen was located in cell plasma. This recombinant protein showed specific immunogenicity.
