Gene cloning, expression, purification and antigenicity analysis of 14 KDa protein of mycobacterium tuberculosis

[Objective] To Clone, express, purify 14 KDa protein of mycobacterium tuberculosis, study its immunological characteristics, and evaluate its potenial value as serological diagnostic reagent of tuberculosis. [Methods] The Gene Coding 14 KDa protein was amplified by polymerase chain reaction (PCR). The gene was inserted with an high-exprssion vector PET-22 b(+) to construct the recombinant plasmid PET-22 b(+)/14 KDa. The recombinant plasmid was transformed into expressive strain E.coli BL21(DE3). The E.coli BL21(DE3) carrying recombinant plasmid was induced with IPTG. The present form of the recombinant protein in expressive strain was analyzed by SDS-PAGE. The recombinant protein was purified by Nickel affinity chromatography, its immunological characteristics was analyzed by Western blotting and ELISA technology. [Results] The clone was analyzed at the nucleotide lever and showed the same DNA sequence coding for natural 14 KDa protein. The recombinant protein expressed in solubility in E.coli BL21(DE3). The expressed protein accounted for 40.8% of total bacteria protein. The purity of terget protein was 94.3% by Nickel affinity chromatography, Western blotting assays indicated that the recombinant protein had satisfactory antigenicity. 14 KDa protein detected TB postive and negative refference serumbased on the mechanism of indirect ELISA. Results showed that the sensitivity was 75.6% and the specificity reached to 96%. [Conclusions] The recombinant protein express of solubility in E.coli BL21(DE3) and have satisfactory antigenicity, and may become one of the immunodiagnostic reagents.