Construction Of Antituberculosis Divalent DNA Vaccine Of PIRES-Ag85A-ESAT6and Its Immunological Effect

Objectives Plasmid pIRES was selected as vector to construct divalent tuberculosis DNA vaccine, which can express Ag85A and ESAT6simultaneously and individually. And the immunogenicity and immunological effect of the vaccine were evaluated in vivo, in order to provide new ideas and experiments basis for the development of new tuberculosis vaccines.Methods1. According to the feature of full-length sequences and restriction sites in the plasmid pTRES reportedby the GeneBank(Ag85A and ESAT6gene) for designing the specific primers through DNAClub and Premier Primer5. Standard strains of mycobacterium tuberculosis H37Rv in the DNA as a template to amplify the Ag85A and ESAT6gene by PCR, The restriction endonucleases of Nhe I,EcoR land Sal I,Eag I were used to digest the amplification products and empty plasmid pIRES for constructing the recombinant plasmid pIRES-Ag85A-ESAT6by ligase. They were detected by bacteria PCR, plasmid digested and plasmid.2. After constructed successfully, the recombinant plasmid of pIRES-Ag85A-ESAT6translated into HD5a screening recombinant plasmid and the plasmid small Plasmid Purification Kit provides DNA.3. After from24to48hours trained, the recombinant plasmidwhich transfected into RAW264.7in5%CO2incubator at37℃by the liposome was detected by western blot.4. A large number of the recombinant plasmid were adopted into BALB/c micethree times with each interval of two weeks. The anti-Ag85A and anti-ESAT6antibodyof the mouse serum which were taken after the last immunization of two weeksdetected by ELISA.5. The cytokines(IFN-γ,IL-2,IL-4) of the mouse splenocytes which incubated with the stimulation of Ag85A and ESAT6protein were detected by ELISA.Results1. The pIRES-Ag85A-ESAT6was constructed successfully by PCR, plasmid double digested and DNA gel electrophoresis, the bands of1017bp and288bp were consistent with the theoretical value of Ag85A and ESAT6which successfully constructed further by the sequence identification.2. After the three different concentrations of pIRES-Ag85A-ESAT6recombinant plasmid transfected into RAW264.7,the Ag85A and ESAT6protein could express in the cells which existed the positive correlation on the transfection dose.3. It was found that the anti-Ag85A and anti-ESAT6antibody were significantly higher (P<0.05) while increased the IgG2a antibody of the divalent vaccine group which transfected by thepIRES-Ag85A-ESAT6plasmid in the immunized mice serum by ELISA.4. The IFN-γ and IL-2of thepIRES-Ag85A-ESAT6plasmid group were significantly increased (P<0.05),whereas IL-4was lower with no significant differences.Conclusion1. The pIRES-Ag85A-ESAT6DNA vaccine could express the antigen of Ag85A and ESAT6in the eukaryotic cells independently.2. The pIRES-Ag85A-ESAT6DNA vaccine could induce a strong specific immune response in mice with the advantages of Thl.3. The pIRES-Ag85A-ESAT6DNA vaccine was singleness, but thevaccine of Ag85A or ESAT6had a strong immune response.