Cloning, expression and purification of 10 kd culture filtrared protein (CFP-10) of Mycobacterium tuberculosis

Abstract

Tuberculosis is a well-known infectious disease in human beings and domestic animals since ancient times. Tuberculin skin test as the only indicator of latent infection with

Mycobacterium tuberculosis has a low specificity and sensitivity value. This test also cannot distinguish between tuberculosis infection and Mycobacterium bovis BCG vaccination, or exposure to environmental mycobacteria. Recent identification of the RD1 region of M. tuberculosis provides a new opportunity for the development of novel diagnostic tools. The purpose of this study was to clone and express the 10-kda culture filtrate protein (CFP-10) of M. tuberculosis in soluble form to be applicable for diagnostic purposes. To reach this aim, DNA was extracted from M. tuberculosis H37Rv and CFP-10 gene was then amplified by using specific primers. Specificity of PCR products were confirmed, and then were cloned into the pET102/D vector. After sequencing and confirming the insertion of desired PCR product into expression vector, recombinant plasmid was initially transformed into Escherichia coli TOP10 and was subsequently transformed into E. coli BL-21 to an expression recombinant protein. Recombinant CFP-10 was purified from the soluble supernatant by metal affinity chromatography. SDS-PAGE analysis was performed to confirm expression of CFP-10 as 28 kDa fusion protein. In this study, we cloned, expressed and purified sufficient amounts of CFP-10 that could be usable in sero-diagnostic tests in future.