The Basic Resaerch In Co-Expression DNA Vaccine Of Tuberculosis Antigen 85A And GM-CSF

Construction, identification and protein expression of an eukaryotic co-expression vector pI85AGMAbstract: Objective: To construct, identify and express an eukaryotic co-expression recombinant containing mouse Granulocyte-macrophage colony stimulating factor (GM-CSF) and tuberculosis antigen 85A (TB Ag85A). Methods: The gene of Ag85A was amplified from plasmid pBSby5 by PCR and cloned directly into plasmid pIRES, and formed recombinant pI85A. GM-CSF was amplified from plasmid pC-GM-CSF by PCR and cloned directly into recombinant pI85A again, and formed co-expression recombinant pI85AGM finally. Results: The eukaryotic expression recombinant plasmid containing GM-CSF and tuberculosis Ag85A had been constructed correctly by identification of restriction enzyme digestion and DNA sequencing. The protein expression of recombinant pI85AGM had been demonstrated correctly by RT-PCR and Western-blotting. Conclusion: An eukaryotic co-expression recombinant plasmid pI85AGM has been constructed successfully. This study provides the possibility of further researches on the development of new type anti-tuberculosis vaccine..Study on immune effect of gene vaccine constructs of tuberculosis antigen 85A and murine granulocyte-macrophage colony stimulating factor in miceAbstract: Objective:To inquire gene vaccine constructs (pI85AGM) containing tuberculosis antigen 85A (TB Ag85A) and mouse granulocyte-macrophage colony stimulating factor (GM-CSF) its immune effect in mice. Methods: After C57BL/6 mice were inoculated with pI85AGM of 100 μg/each mouse (a total of 300 μg/each mouse in three times respectively). There was a interval of three weeks between the inoculations. The activities of cytotoxic T lymphocytes (CTL), natural killer (NK) and spleen cell proliferation responses to antigen were measured by MTT method in immunized mice respectively. The antibody titer of serum in immunized mice was detected by enzyme linked immunosorbent assay(ELISA). Results: The activities of CTL and spleen cell proliferation responses to antigen in mice immunized with pI85AGM were significantly increased respectively than those of mice immunized with blank plasmid. Although NK activity was a little higher in mice immunized with pI85AGM than that of control group mice, it still was lower than that of mice immunized with GM-CSF alone. pI85AGM can induce specific antibody against Ag85A in mice immunized with pI85AGM,however, its titer of antibody was not high. Conclusion: Gene vaccine constructs pI85AGM has been constructed successfully and can induce mice to develop immune effect .This study provides the possibility of further researches protective function in immunized mice...