Expression, characterization and subcellular localization of the Mycobacterium tuberculosis PPE gene Rv1917c
Expression, characterization and subcellular localization of the Mycobacterium tuberculosis PPE gene Rv1917c
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DOI：
10.1054/tube.2001.0304
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发表时间：
2001-01-01
期刊：
影响因子：
3.2
通讯作者：
Everett, MJ
中科院分区：
文献类型：
--
作者：
Sampson, SL;Lukey, P;Everett, MJ
背景：结核分枝杆菌的 PPE 基因家族被认为具有免疫学意义。其中一个成员Rv1917c在临床分离株中具有高度多态性。目的：表征Rv1917c基因多态性和表达，并确定其编码蛋白的细胞定位和糖基化状态。设计：对Rv1917c串联重复区域进行扩增和测序，以确定基因多态性的分子基础。 RT-PCR分析用于检测结核分枝杆菌H37Rv液体培养物中Rv1917c mRNA的表达。该基因被克隆为 3' 末端绿色荧光蛋白 (GFP) 融合体，位于乙酰胺诱导型启动子的下游，并在耻垢分枝杆菌和牛分枝杆菌 BCG 中表达。表达产物的细胞定位和糖基化状态进行了表征。结果：PCR 和序列数据表明，Rv1917c 内不同数量的串联重复序列有助于基因多态性。 RT-PCR 分析表明，Rv1917c mRNA 在结核分枝杆菌 H37Rv 的液体培养物中表达。通过荧光显微镜和流式细胞术观察重组蛋白在耻垢分枝杆菌和牛分枝杆菌BCG中的表达。通过蛋白质印迹证实了预测大小 (166 kDa) 的蛋白质。细胞分级分离研究表明，重组蛋白是疏水性的，表明其与细胞壁相关，而来自抗体结合实验的流式细胞术数据表明其是表面暴露的。对表达蛋白的糖基化状态分析未能证明糖基化。结论：Rv1917c mRNA在结核分枝杆菌H37Rv中表达，Rv1917c基因多态性与可变数量的串联重复序列相关。重组 Rv1917c 蛋白表面暴露。 (C) 哈考特出版有限公司
Setting: The PPE gene family of Mycobacterium tuberculosis is thought to be of immunological significance. One member, Rv1917c, is highly polymorphic in clinical isolates.Objective: To characterize Rv1917c gene polymorphism and expression, and to determine the cellular location and glycosylation status of the encoded protein.Design: Tandem repeat regions of Rv1917c were amplified and sequenced to determine the molecular basis for the gene polymorphism. RT-PCR analysis was utilized to detect expression of Rv1917c mRNA in liquid cultures of M. tuberculosis H37Rv. The gene was cloned as a 3'-terminal green fluorescent protein (GFP) fusion, downstream of an acetamide-inducible promoter, and expressed in Mycobacterium smegmatis and Mycobacterium bovis BCG. The expression product was characterized in terms of cellular location and glycosylation status.Results: PCR and sequence data demonstrated that variable numbers of tandem repeats within Rv1917c contribute to gene polymorphism. RT-PCR analysis demonstrated that Rv1917c mRNA is expressed in liquid cultures of M. tuberculosis H37Rv. Expression of the recombinant protein in M. smegmatis and M. bovis BCG was visualized by fluorescence microscopy and flow cytometry. A protein of the predicted size (166 kDa) was confirmed by Western blotting. Cell fractionation studies demonstrated that the recombinant protein is hydrophobic, suggestive of cell wall-association, while flow cytometric data derived from antibody binding experiments suggested that it is surface exposed. Analysis of the glycosylation status of the expressed protein failed to demonstrate glycosylation.Conclusion: Rv1917c mRNA is expressed in M. tuberculosis H37Rv, and Rv1917c gene polymorphism is associated with variable numbers of tandem repeats. The recombinant Rv1917c protein is surface exposed. (C) Harcourt Publishers Ltd.