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Construction of Mycobacterium tuberculosis eukaryotic expression vector fused expression Ag85B-ESAT6 and its immunological properties

AIM: To construct eukaryotic expression vector expressing secreted protein Ag85B ESAT6 of Mycobacterium tuberculosis (MTB), and to investigate the expression in vitro and the production of humoral immunity response immunized with those recombinant plasmids. METHODS: The gene encoding Ag85B and ESAT6 protein were amplified by polymerase chain reaction (PCR) from genome of MTB H37Rv strain, and inserted into cloning vector pGEM T easy. After sequence analyzed, digested by restriction endonuclease and cloned Ag85B ESAT6 into corresponding sites of the eukaryotic expression vector pcDNA3, and named A Z pcDNA3 E F and E Z pcDNA3 A F respectively. Each plasmid was transfected into CHO cells by electroporation, expression was analyzed by RT PCR at the level of mRNA, by indirect immunofluorescence technique (IIFT)at the level of protein.The specific antibody titers in the sera of BALB/c mice immunized with two recombinant plasmid were detected by ELISA. RESULTS: Sequences of Ag85B and ESAT6 by PCR amplified were identical with that of GenBank reported. The specific mRNA of the fused protein could be detected by RT PCR. CHO cell transfected with two recombinant plasmids were both observed fluorescence by IIFT. Recombinant plasmid could induce higher titer specific antibody in BALB/c mice. The sera specific antibody titer of mice immunized with A Z pcDNA3 E F is 1∶1000, and that of mouse immunized with E Z pcDNA3 A F is 1∶1500. CONCLUSION: Secreted protein Ag85B and ESAT6 of MTB was successfully fusion expressed in eukaryotic expression vector. It may be a promising preventive DNA vaccine against TB.