Construction Of DNA Vaccine Of AG85A Gene Of Mycobacterium Tuberculosis And Observation On Immune Response In Mice Induced By Co-Immunization Of AG85A Gene Vaccine And Plasmid Encoding Human Interleukin 12

The situation of tuberculosis remains severe badly all over the world.Furthermore,the emergence of multi-drug resistant M. tuberculosis (MDR-TB) is reaching the record.The only available vaccine currently is Mycobacterium bovis Bacillus Calmette-Gue′rin (BCG).However,the efficacy of BCG against adult pulmonary TB still remains variable,and can not prevent adult pulmonary tuberculosis.DNA vaccine has its particular advantages when compared with traditional vaccines, however the degree of protection gained from DNA vaccination alone is no more than that afforded by BCG vaccination.It is reported that many researchers utilize plasmids that respectively express immunoregulatory factors and protective antigens to co-immunize animals and expect that immunomodulatory effects and adjuvants of cytokines can enhance role of the host's specific immune response, resulting in an effective immune protection.Ag85A ,one component of secreted proteins of mycobacterium tuberculosis and BCG, can induce strong cellular immune response.The development of acquired cellular immunity is critical for the control of M. tuberculosis infection. The key cytokine required for cell-mediated immunity is gamma interferon (IFN-γ), which functions by stimulating infected macrophages to induce phagolysosomal fusion and killing of intracellular bacteria.The heterodimeric cytokine interleukin-12 is critical for the induction of Th1-like CD4 cells and is produced primarily by dendritic cells (DCs).Human and mice lacking the p40 chain of IL-12 or its receptors are highly susceptibility to M. tuberculosis infection。Plasmids expressing IL-12 or IL-18 have been used as adjuvants in several infectious models.Coadministration of plasmids expressing IL-12 and IL-18 increased the IFN-γT-cell response in DNA vaccination to Ag85B, but only plasmids expressing IL-12 increased protective efficacy.In this study ,we made use of eukaryotic expressing plasmids which respectively encoded Mycobacterium tuberculosis Ag85A antigen and human interleukin 12 in order to observe specific cellular immune response in mice.Objective:To construct DNA vaccine based on Ag85A gene of mycobacterium tuberculosis ,and to observe cellular immune response in mice induced by co-immunization of Ag85A gene vaccine and plasmid encoding human interleukin 12.Methods:1.constructing eukaryotic expressing plasmid ,The gene encoding Ag85A mature protein was amplified by polymerase chain reaction (PCR)from genome of mycobacterium tuberculosis H37Rv strain, and was inserted into cloning vector PMD20-T after restriction endonuclease digestion. Gene fragment encoding Ag85A mature protein was correctly inserted into the vector ,which was confirmed by restriction endonuclease digestion and partial nucleotide sequencing ,and then was subcloned to the corresponding sites of eukaryotic expressing vector pcDNA3.1(+). 2. 50C57BL/6N mi ce were randomly divided into following groups:①Ag85A gene vaccine plus plasmid of PORF- hlL-12;②Ag85A gene vaccine;③BCG(positive control) ;④empty vector alone (negative control);⑤PBS(blank group). Mice were immunized intramuscularly in both hind limbs 3 times at the intervals of three weeks or once subcutaneously with 1×106of viable BCG (about 0.3ml per mouse)at the first time of the DNA immunization. At the 28th days after the last immunization, mice were killed, isolated splenocytes. splenocytes cytotoxicity was detected by LDH release assay, The splenocyte proliferation activities were detected by XTT after stimulation of TB-PPD. IFN-γ,IL-2,and IL-4 levels in supernatant of spleno- lymphocyte cultures was measured by EILSA.Results:1.The DNA vaccine of Ag85A gene of mycobacterium tuberculosis was successfully constructed. 2. The co-immunization group can induce stronger Th1 cellular immune response.In supernatant of spleno-lymphocyte cultures,the levels of IFN-γand IL-2 in the co-immunization group were significantly higher than that of group of Ag85A alone, and they were similar to that in BCG group, the level of IL-4 decreased and was lower than that of BCG group. Special splenocyte T cell cytotoxicity and splenocytes proliferation activities were significantly stronger than other groups.Conclusion:eukaryotic expressing plasmid PORF-hIL-12 can significantly reinforce the specific cellular immune response in mice induced by the Ag85A DNA vaccine.