结核分枝杆菌Rv3194c蛋白的亚细胞定位

【目的】Rv3194c基因编码的是结核分枝杆菌的PDZ信号蛋白,本研究探讨该蛋白的亚细胞定位,为其细胞结合蛋白的筛选奠定基础。【方法】从H37Rv基因组中扩增出编码只含有PDZ结构域的tRv3194c (Rv3194c 1–234 aa)的基因片段,在3′端加T2A和EGFP序列,一并插入真核表达载体构建出pcDNA3.1-tRv3194c-T2A-EGFP。将构建好的质粒瞬时转染L929细胞,并共感染重组痘苗病毒vTF7-3,用间接免疫荧光、流式细胞分选以及Western blotting检测融合蛋白的表达以及亚细胞定位。【结果】成功构建出真核表达载体pcDNA3.1-tRv3194c-T2A-EGFP,瞬时转染L929细胞后融合蛋白tRv3194c定位于线粒体膜上,且重组痘苗病毒vTF7-3的感染有助于靶蛋白表达水平的提高。【结论】Rv3194蛋白的PDZ结构域与线粒体外膜相关蛋白结合,为了解该蛋白在细胞内的致病机制提供重要线索。

Subcellular localization of Rv3194c protein from Mycobacterium tuberculosis

Objective] To investigate the expression and subcellular localization of post-synaptic density-95, Drosophilia tumor suppressor protein diskslarge-1, the tight junction protein zonula occludentes 1 signal protein encoded by Rv3194c gene from Mycobacterium tuberculosis, and to serve the identification of the cellular binding proteins of Rv3194c protein.Methods] The gene encoding tRv3194c (Rv3194c 1-234 aa) with T2A and EGFP sequence was cloned by PCR from the genomic H37Rv, then inserted into the eukaryotic expression vector to construct pcDNA3.1-tRv3194c-T2A-EGFP. Indirect immune fluorescence assay, flow cytometry sorting and Western blotting assay were used to observe subcellular localization, when L929 cells were transfected with constructs before infection with the recombinant vaccinia virus vTF7-3 expressing T7 RNA polymerase.Results] The eukaryotic expression vector pcDNA3.1-tRv3194c-T2A-EGFP was constructed correctly. After the transient transfection with the plasmid, localization of fusion protein tRv3194 in mitochondria was observed by immune fluorescence assay. The dramatically enhanced expression level by co-infection with vTF7-3 before transfection was detected by flow cytometry sorting and Western-blotting assay.Conclusion] Post-synaptic density domain in Rv3194c protein can bind to its ligand protein which located in mitochondrial outer membrane, which provides a key clue to understand its pathogenic mechanism in intracellular.