Dou Jun, Chen Jun Song, Wang Jing, Chen Guo Bin, Zhao Feng Shu, Tang Quan, Fang Xue Song, Chu Li Li, Pan Meng
Department of Pathogenic Biology and Immunology, Southeast University School of Basic Medical Science, Nanjing, China.
Cell Mol Immunol. 2005 Feb;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-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.
本研究开发了一种新型结核基因疫苗，其包含小鼠粒细胞巨噬细胞集落刺激因子（mGM-CSF）和一种结核抗原（Ag85A）。分别通过PCR从含Ag85A的pBSby5和pC-mGM-CSF中扩增编码Ag85A和mGM-CSF的基因。然后将这些基因克隆到质粒pIRES的两个不同多克隆位点，形成新型结核基因疫苗构建体pI85AGM。通过脂质体（Lipofectamine™）将pI85AGM质粒转染到7721细胞系后，通过蛋白质印迹法或RT-PCR鉴定Ag85A和GM-CSF蛋白的表达。然后分别用重组pI85AGM、pI85A、pIGM或单独的质粒接种Balb/c小鼠。通过MTT法检测CTL、NK细胞的活性以及Ag85A刺激的脾细胞增殖。通过ELISA检测抗Ag85A的血清抗体。结果表明，Ag85A和GM-CSF蛋白可在7721细胞系中表达，且在接种pI85AGM的小鼠中CTL活性和脾细胞增殖显著增加，表明接种pI85AGM的小鼠可对Ag85A产生特异性免疫反应。本研究可能为开发新型抗结核基因疫苗提供可能性。
