溶藻弧菌Ⅲ型分泌系统效应蛋白HY322基因的克隆和生物信息学分析
|
摘要
本研究从溶藻弧菌中成功克隆获得HY322基因的编码区序列(GenBank:KX245317.1),该基因全长969 bp,可编码322个氨基酸,应用生物信息学的方法和工具对溶藻弧菌HY9901效应蛋白HY322的理化性质、蛋白结构、遗传进化关系和抗原特性等方面进行预测和分析。结果表明:HY322蛋白为不稳定的亲水性蛋白,蛋白呈酸性,无跨膜区,无明显的信号肽;二级结构以α螺旋为主,进化分析显示溶藻弧菌HY9901与哈维氏弧菌(V.harveyi)聚为一支,表明在进化关系上最为接近。HY322序列中包含有一个与鞭毛形成有关的Fli N功能结构域。多种参数预测了HY322可能的B细胞抗原优势表位,分别是第32~33、100~102、138~140、215~216、235~238、246~249区段。利用SWISS-MODEL软件,得到了HY322亚基三维模型。本研究从生物信息学角度验证了HY322作为弧菌共同抗原的可行性,为下一步的疫苗研发提供了理论依据。
In this study, the coding sequence(Gen Bank: KX245317.1) of HY322 gene was cloned from Vibrio alginolyticus. The total length of its gene was 969 bp, encoding 322 amino acids. The physicochemical properties,protein structure, genetic evolutionary relationship and antigenic characteristics of the effector protein HY322 of vibrio alginolyticus HY9901 were predicted and analyzed by bioinformatics methods and tools. The results showed that HY322 protein might be an unstable hydrophilic and acidic protein without a transmembrane region or a signal peptide, and the secondary structure was mainly α-helix. The evolutionary analysis showed that Vibrio alginolyticus HY9901 and V. harveyi were clustered together, which indicated that their genetic relationship was the closest. The HY322 sequence contained a Fli N superfamily conserved domain associated with Flagellar motor switch. Various parameters predicted that the B-cell preponderant epitopes of HY322 might be localized in the regions of 32~33, 100~102, 138~140, 215~216, 235~238, 246~249. The 3 D structure model of HY322 subunit was simulated by SWISS-MODEL software. This study verified the feasibility of HY322 as a common antigen of Vibrio from the perspective of bioinformatics, which might provide a theoretical basis for the further research and development of vaccine.
In this study, the coding sequence(Gen Bank: KX245317.1) of HY322 gene was cloned from Vibrio alginolyticus. The total length of its gene was 969 bp, encoding 322 amino acids. The physicochemical properties,protein structure, genetic evolutionary relationship and antigenic characteristics of the effector protein HY322 of vibrio alginolyticus HY9901 were predicted and analyzed by bioinformatics methods and tools. The results showed that HY322 protein might be an unstable hydrophilic and acidic protein without a transmembrane region or a signal peptide, and the secondary structure was mainly α-helix. The evolutionary analysis showed that Vibrio alginolyticus HY9901 and V. harveyi were clustered together, which indicated that their genetic relationship was the closest. The HY322 sequence contained a Fli N superfamily conserved domain associated with Flagellar motor switch. Various parameters predicted that the B-cell preponderant epitopes of HY322 might be localized in the regions of 32~33, 100~102, 138~140, 215~216, 235~238, 246~249. The 3 D structure model of HY322 subunit was simulated by SWISS-MODEL software. This study verified the feasibility of HY322 as a common antigen of Vibrio from the perspective of bioinformatics, which might provide a theoretical basis for the further research and development of vaccine.
引文
Ahmed R.,Rafiquzaman S.M.,Hossain M.T.,Lee J.M.,and Kong I.S.,2016,Species-specific detection of Vibrio alginolyticus in shellfish and shrimp by real-time pcr using the groel gene,Aquaculture International,24(1):1-14
Burdette D.L.,and Seemann J.K.,2009,Vibrio vopq induces pi3-kinase-independent autophagy and antagonizes phagocytosis,Molecular Microbiology,73(4):639-649
Bzymek K.P.,Hamaoka B.Y.,and Ghosh P.,2012,Two translation products of yersinia yscq assemble to form a complex essential to type iii secretion,Biochemistry,51(8):1669-1677
Heo Y.J.,Lee C.H.,Baek M.S.,Ahn H.M.,Hwang Y.S.,and Park K.H.,2012,Morphological characterization of Vibrio alginolyticus specific bacteriophage isolated from fish farms on west coast of Korea,Journal of Fish Pathology,25(3):165-172
Hunter T.,2000,Signaling--2000 and beyond,Cell,100(1):113-127
Kogure K.,and Ikemoto E.H.,1998,Attachment of Vibrio alginol yticus to glass surfaces is dependent on swimming speed,Journal of Bacteriology,180(4):932-937
Khan I.H.,Reese T.S.,and Khan S.,1992,The cytoplasmic component of the bacterial flagellar motor,Proceedings of the National Academy of Sciences of the United States of America,89(13):5956-5960
Kim J.H.,Lee J.,Oh B.,K imm K.,and Koh I.,2004,Prediction of phosphorylation sites using svms,Bioinformatics,20(17):3179-3184
Murzin A.G.,Brenner S.E.,Hubbard T.,and Chothia C.,1995,SCOP:a structural classification of proteins for the investigation of sequences and structures,Journal of Molecular Biology,247(4):536-540
Pang H.Y.,Zhou Z.J.,Ding Y.,Huang Y.C.,Wu Z.H.,and Jian J.C.,2014a,Molecular cloning and bioinformatics analysis of T3SS chaperone escort protein Vsc O from Vibrio alginolyticus,Shengwu Jishu Tongbao(Biotechnology Bulletin),27(6):155-161(庞欢瑛,周泽军,丁燏,黄郁葱,吴灶和,简纪常,2014a,溶藻弧菌Ⅲ型分泌系统分子伴侣护航蛋白Vsc O的基因克隆及其生物信息学分析,生物技术通报,27(6):155-161)
Pang H.Y.,Zhou Z.J.,Ding Y.,Huang Y.C.,Wu Z.H.,and Jian J.C.,2014b,Prokaryotic expression and immunogenicity of the typeⅢsecretion system injectisome protein Vsc O from Vibrio alginolyticus,Guangdong Haiyang Daxue Xuebao(Journal of Guangdong Ocean University),5(3):261-269(庞欢瑛,周泽军,丁燏,黄郁葱,吴灶和,简纪常,2014b,溶藻弧菌Ⅲ型分泌系统注射装置蛋白Vsc O的原核表达及免疫原性,广东海洋大学学报,5(3):261-269)
Snoussi M.,Noumi E.,Hajlaoui H.,Usai D.,Sechi L.A.,Zanetti S.,and Bakhrouf A.,2009,High potential of adhesion to abiotic and biotic materials in fish aquaculture facility by Vibrio alginolyticus strains,Journal of Applied Microbiology,106(5):1591-1599
Tian M.,Chen X.,Xiong Q.,Xiong J.,Xiao C.,Ge F.,Yang F.,and Miao W.,2014,Phosphoproteomic analysis of protein phosphorylation networks in tetrahymena thermophila,a model single-celled organism,Molecular&Cellular Proteomics,13(2):503-519
Wilkins M.R.,Gasteiger E.,Bairoch A.,Sanchez J.C.,Williams K.L.,Appel R.D.,and Hochstrasser D.F.,1999,Protein identification and analysis tools in the expasy server,Methods in Molecular Biology,112:531-552
Xie H.X.,Yu H.B.,Zheng J.,Nie P.,Foster L.J.,Mok Y.K.,Finlay B.B.,and Leung K.Y.,2010,Eseg,an effector of the type iii secretion system of edwardsiella tarda,triggers microtubule destabilization,Infection&Immunity,78(12):5011-5021
Zhao R.,Pathak N.,Jaffe H.,Reese T.S.,and Khan S.,1996,Flin is a major structural protein of the c-ring in the salmonella typhimurium flagellar basal body,Journal of Molecular Biology,261(2):195-208
Burdette D.L.,and Seemann J.K.,2009,Vibrio vopq induces pi3-kinase-independent autophagy and antagonizes phagocytosis,Molecular Microbiology,73(4):639-649
Bzymek K.P.,Hamaoka B.Y.,and Ghosh P.,2012,Two translation products of yersinia yscq assemble to form a complex essential to type iii secretion,Biochemistry,51(8):1669-1677
Heo Y.J.,Lee C.H.,Baek M.S.,Ahn H.M.,Hwang Y.S.,and Park K.H.,2012,Morphological characterization of Vibrio alginolyticus specific bacteriophage isolated from fish farms on west coast of Korea,Journal of Fish Pathology,25(3):165-172
Hunter T.,2000,Signaling--2000 and beyond,Cell,100(1):113-127
Kogure K.,and Ikemoto E.H.,1998,Attachment of Vibrio alginol yticus to glass surfaces is dependent on swimming speed,Journal of Bacteriology,180(4):932-937
Khan I.H.,Reese T.S.,and Khan S.,1992,The cytoplasmic component of the bacterial flagellar motor,Proceedings of the National Academy of Sciences of the United States of America,89(13):5956-5960
Kim J.H.,Lee J.,Oh B.,K imm K.,and Koh I.,2004,Prediction of phosphorylation sites using svms,Bioinformatics,20(17):3179-3184
Murzin A.G.,Brenner S.E.,Hubbard T.,and Chothia C.,1995,SCOP:a structural classification of proteins for the investigation of sequences and structures,Journal of Molecular Biology,247(4):536-540
Pang H.Y.,Zhou Z.J.,Ding Y.,Huang Y.C.,Wu Z.H.,and Jian J.C.,2014a,Molecular cloning and bioinformatics analysis of T3SS chaperone escort protein Vsc O from Vibrio alginolyticus,Shengwu Jishu Tongbao(Biotechnology Bulletin),27(6):155-161(庞欢瑛,周泽军,丁燏,黄郁葱,吴灶和,简纪常,2014a,溶藻弧菌Ⅲ型分泌系统分子伴侣护航蛋白Vsc O的基因克隆及其生物信息学分析,生物技术通报,27(6):155-161)
Pang H.Y.,Zhou Z.J.,Ding Y.,Huang Y.C.,Wu Z.H.,and Jian J.C.,2014b,Prokaryotic expression and immunogenicity of the typeⅢsecretion system injectisome protein Vsc O from Vibrio alginolyticus,Guangdong Haiyang Daxue Xuebao(Journal of Guangdong Ocean University),5(3):261-269(庞欢瑛,周泽军,丁燏,黄郁葱,吴灶和,简纪常,2014b,溶藻弧菌Ⅲ型分泌系统注射装置蛋白Vsc O的原核表达及免疫原性,广东海洋大学学报,5(3):261-269)
Snoussi M.,Noumi E.,Hajlaoui H.,Usai D.,Sechi L.A.,Zanetti S.,and Bakhrouf A.,2009,High potential of adhesion to abiotic and biotic materials in fish aquaculture facility by Vibrio alginolyticus strains,Journal of Applied Microbiology,106(5):1591-1599
Tian M.,Chen X.,Xiong Q.,Xiong J.,Xiao C.,Ge F.,Yang F.,and Miao W.,2014,Phosphoproteomic analysis of protein phosphorylation networks in tetrahymena thermophila,a model single-celled organism,Molecular&Cellular Proteomics,13(2):503-519
Wilkins M.R.,Gasteiger E.,Bairoch A.,Sanchez J.C.,Williams K.L.,Appel R.D.,and Hochstrasser D.F.,1999,Protein identification and analysis tools in the expasy server,Methods in Molecular Biology,112:531-552
Xie H.X.,Yu H.B.,Zheng J.,Nie P.,Foster L.J.,Mok Y.K.,Finlay B.B.,and Leung K.Y.,2010,Eseg,an effector of the type iii secretion system of edwardsiella tarda,triggers microtubule destabilization,Infection&Immunity,78(12):5011-5021
Zhao R.,Pathak N.,Jaffe H.,Reese T.S.,and Khan S.,1996,Flin is a major structural protein of the c-ring in the salmonella typhimurium flagellar basal body,Journal of Molecular Biology,261(2):195-208