结核分枝杆菌Rv3194c蛋白的亚细胞定位
|
摘要
【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。
[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.
[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.
引文
[1]O’Reilly LM,Daborn CJ.The epidemiology of Mycobacterium bovis infections in animals and man:a review.Tubercle and Lung Disease,1995,76 Suppl:1-46.
[2]Someya S,Hayashi O,Tajima Y,Endo M.Studies on the virulence of tubercle bacilli.III.The fate of human type and bovine type tubercle bacille in various organs of intravenously infected mice.Kekkaku,1951,26(2):74-78.
[3]Ascoli A.Reaction of the cholemic calf to Mycobacterium tuberculosis,bovine type.Giornale Italiano Della Tubercolosi,1950,4(3):129-130.
[4]Castets M,Boisvert H,Grumbach F,Brunel M,Rist N.Tuberculosis bacilli of the African type:preliminary note.Revue de Tuberculose et de Pneumologie,1968,32(2):179-184.
[5]Flynn JL,Chan J.Immunology of tuberculosis.Annual Review of Immunology,2001,19(1):93-129.
[6]van Crevel R,Ottenhoff THM,van der Meer JWM.Innate Immunity to Mycobacterium tuberculosis.Clinical Microbiology Reviews,2002,15(2):294-309.
[7]Puvaci? S,Dizdarevi?J,Santi?Z,Mulaomerovi?M.Protective effect of neonatal BCG vaccines against tuberculous meningitis.Bosnian Journal of Basic Medical Sciences,2004,4(1):46-49.
[8]Shenai S,Rodrigues C,Mehta A.Rapid speciation of 15clinically relevant mycobacteria with simultaneous detection of resistance to rifampin,isoniazid,and streptomycin in Mycobacterium tuberculosis complex.International Journal of Infectious Diseases,2009,13(1):46-58.
[9]Javier RT,Rice AP.Emerging theme:cellular PDZ proteins as common targets of pathogenic viruses.Journal of Virology,2011,85(22):11544-11556.
[10]Hormozdiari F,Salari R,Bafna V,Sahinalp SC.Protein-protein interaction network evaluation for identifying potential drug targets.Journal of Computational Biology,2010,17(5):669-684.
[11]Houslay MD.Disrupting specific PDZ domain-mediated interactions for therapeutic benefit.British Journal of Pharmacology,2009,158(2):483-485.
[12]Zhao DY,Lin LL,Wen FL.Expression,purification and characterization of Rv3194c protein from Mycobacterium tuberculosis.Acta Microbiologica Sinica,2016,56(12):1847-1855.(in Chinese)赵东岳,林莉莉,温福利.结核分枝杆菌Rv3194c蛋白的表达、纯化及活性鉴定.微生物学报,2016,56(12):1847-1855.
[13]Xiao XD,Chakraborti S,Dimitrov AS,Gramatikoff K,Dimitrov DS.The SARS-CoV S glycoprotein:expression and functional characterization.Biochemical and Biophysical Research Communications,2003,312(4):1159-1164.
[14]Simmons G,Reeves JD,Rennekamp AJ,Amberg SM,Piefer AJ,Bates P.Characterization of severe acute respiratory syndrome-associated coronavirus(SARS-CoV)spike glycoprotein-mediated viral entry.Proceedings of the National Academy of Sciences of the United States of America,2004,101(12):4240-4245.
[15]Wu YS,Feng Y,Dong WQ,Zhang YM,Li M.A vaccinia replication system for producing recombinant hepatitis Cvirus.World Journal of Gastroenterology,2004,10(18):2670-2674.
[2]Someya S,Hayashi O,Tajima Y,Endo M.Studies on the virulence of tubercle bacilli.III.The fate of human type and bovine type tubercle bacille in various organs of intravenously infected mice.Kekkaku,1951,26(2):74-78.
[3]Ascoli A.Reaction of the cholemic calf to Mycobacterium tuberculosis,bovine type.Giornale Italiano Della Tubercolosi,1950,4(3):129-130.
[4]Castets M,Boisvert H,Grumbach F,Brunel M,Rist N.Tuberculosis bacilli of the African type:preliminary note.Revue de Tuberculose et de Pneumologie,1968,32(2):179-184.
[5]Flynn JL,Chan J.Immunology of tuberculosis.Annual Review of Immunology,2001,19(1):93-129.
[6]van Crevel R,Ottenhoff THM,van der Meer JWM.Innate Immunity to Mycobacterium tuberculosis.Clinical Microbiology Reviews,2002,15(2):294-309.
[7]Puvaci? S,Dizdarevi?J,Santi?Z,Mulaomerovi?M.Protective effect of neonatal BCG vaccines against tuberculous meningitis.Bosnian Journal of Basic Medical Sciences,2004,4(1):46-49.
[8]Shenai S,Rodrigues C,Mehta A.Rapid speciation of 15clinically relevant mycobacteria with simultaneous detection of resistance to rifampin,isoniazid,and streptomycin in Mycobacterium tuberculosis complex.International Journal of Infectious Diseases,2009,13(1):46-58.
[9]Javier RT,Rice AP.Emerging theme:cellular PDZ proteins as common targets of pathogenic viruses.Journal of Virology,2011,85(22):11544-11556.
[10]Hormozdiari F,Salari R,Bafna V,Sahinalp SC.Protein-protein interaction network evaluation for identifying potential drug targets.Journal of Computational Biology,2010,17(5):669-684.
[11]Houslay MD.Disrupting specific PDZ domain-mediated interactions for therapeutic benefit.British Journal of Pharmacology,2009,158(2):483-485.
[12]Zhao DY,Lin LL,Wen FL.Expression,purification and characterization of Rv3194c protein from Mycobacterium tuberculosis.Acta Microbiologica Sinica,2016,56(12):1847-1855.(in Chinese)赵东岳,林莉莉,温福利.结核分枝杆菌Rv3194c蛋白的表达、纯化及活性鉴定.微生物学报,2016,56(12):1847-1855.
[13]Xiao XD,Chakraborti S,Dimitrov AS,Gramatikoff K,Dimitrov DS.The SARS-CoV S glycoprotein:expression and functional characterization.Biochemical and Biophysical Research Communications,2003,312(4):1159-1164.
[14]Simmons G,Reeves JD,Rennekamp AJ,Amberg SM,Piefer AJ,Bates P.Characterization of severe acute respiratory syndrome-associated coronavirus(SARS-CoV)spike glycoprotein-mediated viral entry.Proceedings of the National Academy of Sciences of the United States of America,2004,101(12):4240-4245.
[15]Wu YS,Feng Y,Dong WQ,Zhang YM,Li M.A vaccinia replication system for producing recombinant hepatitis Cvirus.World Journal of Gastroenterology,2004,10(18):2670-2674.