Molecular Cloning, Expression and Purification of Protein TB۱۰.۴ Secreted by Mycobacterium Tuberculosis

Objective(s) Tuberculosis (TB) is the leading cause of mortality among the infectious diseases, especially in developing countries. One of the main goals in tuberculosis research is to identify antigens which have the ability of inducing cellular and/or humoral immunity in order to use them in diagnostic reagents or vaccine design. The aim of this study was to clone and express the TB'۰.۴ protein in Escherichia coli expression system. Materials and Methods DNA was extracted from Mycobacterium tuberculosis H۳۷Rv. Gene specific primers were designed using Gene Runner software according to sanger sequence database. Gene tb'۰.۴ fragment was amplified by PCR method and purified tb'۰.۴ gene was cloned into pET ۱۰۲/D vector. Plasmid containing pET۱۰۲/D-۱۰.۴ was transformed into competence E. coli TOP'۰. A positive transformant was chosen and plasmids DNA was isolated and subsequently transformed into competence E. coli BL۲'(DE۳). The bacterium was induced by IPTG and its lysates were loaded directly onto SDS-PAGE. Purified recombinant protein was achieved using metal affinity chromatography (Ni-nitrilotriacetic acid). Results TB'۰.۴ molecule was successfully cloned, expressed, and purified. An approximately ۲۶.۴ kDa exogenous protein was observed on the SDS-PAGE. The recombinant protein was confirmed by DNA sequencing of correct insert. Conclusion The success of expressing the TB'۰.۴ protein could serve as a basis for further studies on the usefulness of the gene and its expression product in the development of subunit vaccine and diagnostic method.