Construction of Mycobacterium tuberculosis eukaryotic expression vector fused expression Ag85Bª²ESAT6 and its immunological properties

    SHI Changª²Hong, FAN Xiongª²Lin, XU Zhiª²Kai, LI Yuan, BAI Yinª²Lan, XUE Ying

    Department of Microbiology, School of Basic Medicine, Fourth Military Medical University, Xi¬ðan 710033, China

    ¡¾Abstract¡¿ AIM: To construct eukaryotic expression vector expressing secreted protein Ag85Bª²ESAT6 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 pGEMª²Tª²easy. After sequence analyzed, digested by restriction endonuclease and cloned Ag85Bª²ESAT6 into corresponding sites of the eukaryotic expression vector pcDNA3, and named AZª²pcDNA3ª²EF and EZª²pcDNA3ª²AF respectively. Each plasmid was transfected into CHO cells by electroporation, expression was analyzed by RTª²PCR 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 RTª²PCR. 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 AZª²pcDNA3ª²EF is 1¡Ã1000, and that of mouse immunized with EZª²pcDNA3ª²AF is 1¡Ã1500. 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. ......