Subcellular localization of Rv3194c protein from Mycobacterium tuberculosis

Abstract:

[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.