Characterisation of Toxoplasma gondii engineered to express mouse interferon-gamma
Characterisation of Toxoplasma gondii engineered to express mouse interferon-gamma
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DOI：
10.1016/s0020-7519(03)00204-2
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发表时间：
2003-11-01
影响因子：
4
通讯作者：
Nagasawa, H
中科院分区：
文献类型：
--
作者：
Nishikawa, Y;Xuan, XN;Nagasawa, H
近年来的研究表明，利用弓形虫作为外源蛋白表达系统是可行的。为了更好地了解γ-干扰素（IFN-γ）依赖性弓形虫免疫的机制，将IFN-γ基因稳定转染弓形虫，在GRA 1启动子的控制下。免疫荧光分析显示，重组小鼠IFN-γ定位于与致密颗粒一致的离散点状结构，并分泌到空泡空间中。IFN-γ的产生在细胞外寄生虫和寄生虫感染的细胞中都是可检测的。重组寄生虫的生长在小鼠巨噬细胞系（J774A.1细胞）中受到抑制，但在猴肾粘附成纤维细胞（Vero细胞）中不受抑制，证明IFN-γ的种属特异性。添加抗小鼠IFN-γ抗体导致重组寄生虫的生长恢复，表明从寄生的宿主细胞分泌的IFN-γ穿过寄生虫空泡膜，以旁分泌方式起作用。逆转录（RT）-PCR分析显示，诱导型一氧化氮合酶的mRNA和重组寄生虫感染的J774A.1细胞中的一氧化氮生产的高水平的显着表达。L-精氨酸依赖性效应途径的竞争性抑制剂N-G-单甲基-L-精氨酸抑制了J774A.1细胞中重组寄生虫生长的减少。综上所述，我们的数据表明，T.弓形虫表达系统可能为细胞因子基因表达提供新的工具，并且可以合理地设计表达细胞因子基因的工程寄生虫用于对弓形虫的免疫应答的研究。(C)2003年澳大利亚寄生虫学会由Elsevier Ltd.出版。保留所有权利。
Recent studies have shown the feasibility of using Toxoplasma gondii as an expression system for heterologous protein. For better understanding of the mechanism of interferon-gamma (IFN-gamma) dependent immunity to T gondii, the parasites were stably transfected with IFN-gamma gene, under control of the GRA1 promoter. Immunofluorescence analyses showed that recombinant mouse IFN-gamma localised to discrete punctuate structures consistent with dense granules and secreted into the vacuolar space. The production of IFN-gamma was detectable in both extracellular parasites and the parasite-infected cells. Growth of the recombinant parasites was inhibited in the mouse macrophage cell line (J774A.1 cells), but not in monkey kidney adherent fibroblasts (Vero cells), demonstrating the species-specificity of IFN-gamma. Addition of anti-mouse IFN-gamma antibody resulted in growth recovery of the recombinant parasites, suggesting that IFN-gamma, secreted from the parasitised host cells across the parasitophorous vacuole membrane, acted in a paracrine manner. Reverse transcription (RT)-PCR analysis revealed significant expression of inducible nitric oxide synthase mRNA and high levels of nitric oxide production in recombinant parasite-infected J774A.1 cells. A competitive inhibitor of the L-arginine-dependent effector pathway, N-G-monomethyl-L-arginine, inhibited the reduction of recombinant parasite growth in J774A.1 cells. Taken together, our data suggest that the T. gondii expression system may provide a new tool for cytokine gene expression and that parasites engineered to express a cytokine gene may be rationally designed for use in studies on immune responses to T gondii. (C) 2003 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.