Isolation, Cloning and Molecular Analysis of Ag85a and Tb10.4 Genes From Mycobacterium Tuberculosis

Background: novel tuberculosis (tb) vaccines that aim to boost and/or replace bacillus calmette-guerin (bcg) are currently in development. dna vaccines can stimulate both humoral and cell-mediated immunity in different animal models of tb and is thought to be a promising strategy in the development of new vaccines against tb. the aim of this study was to design and construct a dna vaccine encoding ag85a and tb10.4 fusion genes of mycobacterium tuberculosis. materials and methods: tb10.4 fragment was amplified by pcr and the product was digested with restriction enzymes. next, it was cloned into the pcdna3.1+ plasmid. the ag85a gene and pcdna3.1+/tb10.4 plasmid were digested by ecori and bamhi restriction enzymes. constructed vector was sequenced. the molecular analysis was done using bioinformatics software. new chimeric vector containing ag85a-tb10.4 genes were purified. expression of pcdna3.1+/tb10.4-ag85a plasmid was confirmed in eukaryotic cells. results: fragments of 297 bp for tb10.4 and 1017 bp for ag85a were observed in agarose gel electrophoresis. alignment of ag85a-tb10.4 genome sequence with reference genes in genbank showed exact identities that indicate correction of all cloning procedures. transfection of eukaryotic cells with pcdna3.1+/tb10.4-ag85a vector and existence of tb10.4-ag85a fusion gene were both confirmed with rt-pcr. conclusion: in this study, tb10.4 and ag85a genes were isolated from mycobacterium tuberculosis h37rv strain and cloned into pcdna3.1+. also, the capability of constructed vector in producing fusion ag85a-tb10.4 protein was confirmed with rt-pcr. pcdna3.1+/tb10.4-ag85a vector can be used for further studies in future.