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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-CSFwere amplified by PCR respectively from the Ag85A-containing pBSby5 and pC-mGM-CSF.The genes were thencloned 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 andGM-CSF proteins was identified by Western blotting or RT-PCR.Then Balb/c mice were inoculated with therecombinant pI85AGM,pI85A,pIGM or plasmid alone,respectively.The activities of CTL,NK cells and theAg85A-stimulated proliferation of spleen cells were measured by MTT method.The serum antibody against Ag85Awas detected by ELISA.The results showed that the Ag85A and GM-CSF proteins could be expressed in 7721 cellline and the activity of CTLs and the proliferation of spleen cells were significantly increased in thepI85AGM-immunized mice,indicating that the pI85AGM-immunized mice could generate specific immuneresponses 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.
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- CSFwere 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 lines by Lipofectamine ™, the expression of Ag85A andGM-CSF proteins were identified by Western blotting or RT-PCR. Balb / c mice were inoculated with therecombinant pI85AGM, pI85A, pIGM or plasmid alone, respectively. These activities of CTL, NK cells and theAg85A-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 cellline and the activity of CTLs and the proliferation of spleen ce lls 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 potential for developing novel anti-TB gene vaccine. Cellular & Molecular Immunology. 2005; 2 (1): 57 -62. Cellular & Molecular Immunology. 2005; 2 (1): 57-62.