Molecular cloning of alpha antigen like protein gene of Mycobacterium leprae and its over production in Escherichia coli

I have constructed the genomic library of M. leprae Thai 53 strain, and cloned the alpha antigen like protein gene by plaque hybridization method by using M. leprae alpha antigen DNA fragment as probe which was characterized in the previous study, I have termed it as alpha 2 antigen gene. The alpha 2 antigen gene has been characterized by sequencing. By comparing the deduced amino acid sequence of alpha and alpha 2 antigen with 85 complex antigen of other mycobacteria. I have found the higher homology between alpha 2 antigen and 85A antigen and between alpha antigen and 85B antigen. We have constructed the over expression system of M. leprae alpha and alpha 2 antigen gene in E. coli using vector pMALc-RI. Recombinant alpha and alpha 2 antigen has been purified by amylose column chromatography at the purity of more than 95%. More than 6 mg and more than 10 mg of recombinant alpha and alpha 2 antigen has been obtained from 200 ml of liquid culture, respectively. ELISA tests have been performed with the sera of leprosy patient and healthy control against the recombinant alpha and alpha 2 antigens. The antibody titers in sera of leprosy patient against the two kinds of antigens were all much higher than healthy controls. The antibody titer against the alpha 2 antigen was higher than that against alpha antigen. Recombinant alpha and alpha 2 antigens in this study could be used as a new specific antigen for serodiagnosis of leprosy.