A novel tuberculosis(TB)gene vaccine containing mouse granulocyte macrophage-colony stimulating factor (mGM-CSF)and a TB antigen(Ag85A)was developed in this study.The genes encoding Ag85A and mGM-CSF were amplified by...A novel tuberculosis(TB)gene vaccine containing mouse granulocyte macrophage-colony stimulating factor (mGM-CSF)and a TB antigen(Ag85A)was developed in this study.The genes encoding Ag85A and mGM-CSF were amplified by PCR respectively from the Ag85A-containing pBSby5 and pC-mGM-CSF.The genes were then cloned into two different polylinker sites of plasmid pIRES,forming a novel TB gene vaccine construct pI85AGM. Following transfection of pI85AGM plasmid into 7721 cell line by Lipofectamine^(TM),the expression of Ag85A and GM-CSF proteins was identified by Western blotting or RT-PCR.Then Balb/c mice were inoculated with the recombinant pI85AGM,pI85A,pIGM or plasmid alone,respectively.The activities of CTL,NK cells and the Ag85A-stimulated proliferation of spleen cells were measured by MTT method.The serum antibody against Ag85A was detected by ELISA.The results showed that the Ag85A and GM-CSF proteins could be expressed in 7721 cell line and the activity of CTLs and the proliferation of spleen cells were significantly increased in the pI85AGM-immunized mice,indicating that the pI85AGM-immunized mice could generate specific immune responses to Ag85A.This study might provide possibility for developing novel anti-TB gene vaccine.Cellular & Molecular Immunology.2005;2(1):57-62.Cellular & Molecular Immunology.2005;2(1):57-62.展开更多
文摘A novel tuberculosis(TB)gene vaccine containing mouse granulocyte macrophage-colony stimulating factor (mGM-CSF)and a TB antigen(Ag85A)was developed in this study.The genes encoding Ag85A and mGM-CSF were amplified by PCR respectively from the Ag85A-containing pBSby5 and pC-mGM-CSF.The genes were then cloned into two different polylinker sites of plasmid pIRES,forming a novel TB gene vaccine construct pI85AGM. Following transfection of pI85AGM plasmid into 7721 cell line by Lipofectamine^(TM),the expression of Ag85A and GM-CSF proteins was identified by Western blotting or RT-PCR.Then Balb/c mice were inoculated with the recombinant pI85AGM,pI85A,pIGM or plasmid alone,respectively.The activities of CTL,NK cells and the Ag85A-stimulated proliferation of spleen cells were measured by MTT method.The serum antibody against Ag85A was detected by ELISA.The results showed that the Ag85A and GM-CSF proteins could be expressed in 7721 cell line and the activity of CTLs and the proliferation of spleen cells were significantly increased in the pI85AGM-immunized mice,indicating that the pI85AGM-immunized mice could generate specific immune responses to Ag85A.This study might provide possibility for developing novel anti-TB gene vaccine.Cellular & Molecular Immunology.2005;2(1):57-62.Cellular & Molecular Immunology.2005;2(1):57-62.
