溶藻弧菌Ⅲ型分泌系统分子伴侣护航蛋白VscO的基因克隆及其生物信息学分析

生物技术通报 ›› 2014, Vol. 0 ›› Issue (6): 155-161.

溶藻弧菌Ⅲ型分泌系统分子伴侣护航蛋白VscO的基因克隆及其生物信息学分析

庞欢瑛^1，2，3, 周泽军^1，2，3, 丁燏^1，2，3, 黄郁葱^1，2，3, 吴灶和^2，3，4, 简纪常^1，2，3

1.广东海洋大学水产学院，湛江 524088；
2.广东省水产经济动物病原生物学及流行病学重点实验室，湛江 524088
3.广东省教育厅水产经济动物病害控制重点实验室，湛江 524088
4.仲恺农业工程学院，广州 510225

收稿日期:2014-04-16 出版日期:2014-06-25 发布日期:2014-06-25
作者简介:庞欢瑛，女，博士，讲师，研究方向：水产经济动物病害；E-mail：phying1218@163.com
基金资助:
国家科技支撑计划（2012BAD17B02），广东省自然科学基金项目（S2013040014562）

Molecular Cloning and Bioinformatics Analysis of T3SS Chaperone Escort Protein VscO from Vibrio alginolyticus

Pang Huanying^1，2，3,Zhou Zejun^1，2，3,Ding Yu ^1，2，3,Huan Yucong^1，2，3,Wu Zaohe^2，3，4,Jian Jichang^1，2，3

1. Fisheries College of Guangdong Ocean University，Zhanjiang 524088
2. Guangdong Provincial Key Laboratory of Pathogenic Biology and Epidemiology for Aquatic Economic Animals，Zhanjiang 524088
3. Key Laboratory of Diseases Controlling for Aquatic Economic Animals of Guangdong Higher Education Institutions，Zhanjiang 524088
4. Zhongkai University of Agriculture and Engineering，Guangzhou 510225

Received:2014-04-16 Published:2014-06-25 Online:2014-06-25

摘要/Abstract

摘要：

克隆了溶藻弧菌（Vibrio alginolyticus）ZJ03株Ⅲ型分泌系统（T3SS）分子伴侣护航蛋白（Chaperone escort protein）vscO基因，并对其进行生物信息学分析。结果表明，vscO基因全长462 bp，编码153个氨基酸，理论分子量约为18.43 kD，pI值为9.22。细胞定位分析显示vscO基因位于外周质，不存在信号肽，无跨膜区。vscO基因具有转录起始区-35区和-10区，以及翻译识别信号SD序列（Shine-Dalgarno sequence）。该氨基酸序列含有多种活性位点，如蛋白激酶C磷酸化位点等。系统进化树发现溶藻弧菌的VscO蛋白与副溶血弧菌聚为同一亚族。VscO亚基三维结构模型显示其与副溶血弧菌T3SS 的YscO蛋白有相似构型。信号通路分析推测VscO位于T3SS的针状样结构上。蛋白网络互作图谱发现VscO与10种T3SS蛋白具有相邻关系。本研究结果将为溶藻弧菌Ⅲ型分泌系统护航机制的研究奠定基础。

关键词: 溶藻弧菌, Ⅲ型分泌系统（T3SS）, 分子伴侣护航, vscO基因

Abstract:

Primers for PCR cloning were designed according to the whole genome sequence of Vibrio alginolyticus published in GenBank. The type III secretion system（T3SS）chaperone escort protein vscO gene of V.alginolyticus strain ZJ03 was amplified by PCR and cloned into pMD18-T vector. Sequence analysis revealed that vscO gene is 462 bp and encodes a putative protein of 153 amino acids. The predicted molecular weight（MW）of VscO was 18.43 kD with an estimated pI of 9.22. Using SignalP 4.0 and TMHMM Server 2.0 software, it was predicted that the VscO protein was located in periplasm. It did not contain a signal peptide or a transmembranous region. The vscO gene with transcription initiation region at -35 and -10 region, and a translation recognition signal sequence（SD Shine-Dalgarno sequence）. This protein had some active sites, such as phosphorylation sites. To further analyze the evolutionary relationship among VscO, a molecular phylogenetic tree was constructed using Mega 5.0 software. In this tree, the VscO protein showed high genetic relationship with Vibrio parahaemolyticus. The three-dimensional structure of VscO was determined using SWISS-MODEL work-space and it had a similar structure with YscO protein of V. parahaemolyticus. Signaling pathway analysis showed that VscO is located at the “needle” site of T3SS. Protein interaction map network found that the relation between VscO and 10 kinds of T3SS proteins was neighbourhood. These results can provide a basis for further studies on the chaperone escort machanism used by T3SS export pathway of Vibrio species.

Key words: Vibrio alginolyticus, T3SS, Chaperone escort, vscO gene

, 庞欢瑛, 周泽军, 丁燏, 黄郁葱, 吴灶和, 简纪常. 溶藻弧菌Ⅲ型分泌系统分子伴侣护航蛋白VscO的基因克隆及其生物信息学分析[J]. 生物技术通报, 2014, 0(6): 155-161.

Pang Huanying,Zhou Zejun,Ding Yu,Huan Yucong,Wu Zaohe,Jian Jichang. Molecular Cloning and Bioinformatics Analysis of T3SS Chaperone Escort Protein VscO from Vibrio alginolyticus[J]. Biotechnology Bulletin, 2014, 0(6): 155-161.

参考文献

[1] Sadok K, Mejdi S, Nourhen S, et al. Phenotypic characterization and RAPD fingerprinting of Vibrio parahaemolyticus and Vibrio alginolyticus isolated during Tunisian fish farm outbreaks[J] . Folia Microbiologica（Praha）, 2013, 58（1）：17-26.
[2] Chang CC, Yeh MS, Lin HK, et al. The effect of Vibrio alginolyticus infection on caspase-3 expression and activity in white shrimp Litopenaeus vannamei[J] . Fish and Shellfish Immunology, 2008, 25（5）：672-678.
[3] Ben Kahla-Nakbi A, Chaieb K, Bakhrouf A. Investigation of several virulence properties among Vibrio alginolyticus strains isolated from diseased cultured fish in Tunisia[J] . Diseases of Aquatic Organisms, 2009, 86（1）：21-28.
[4] Campanelli A, Sanchez-Politta S, Saurat JH. Cutaneous ulceration after an octopus bite：infection due to Vibrio alginolyticus, an emerging pathogen[J] . Ann Dermatol Venereol, 2008, 135（3）：225-227.
[5] Sganga G, Cozza V, Spanu T, et al. Global climate change and wound care：case study of an off-season Vibrio alginolyticus infection in a healthy man[J] .Ostomy Wound Manage, 2009, 55（4）：60-62.
[6] Zhao Z, Zhang L, Ren C, et al. Autophagy is induced by the type III secretion system of Vibrio alginolyticus in several mammalian cell lines[J] . Archives of Microbiology, 2011, 193（1）：53-61.
[7] Caron E, Crepin VF, Simpson N, et al. Subversion of actin dynamics by EPEC and EHEC[J] . Current Opinion in Microbiology, 2006, 9（1）：40-45.
[8] Zhao Z, Chen C, Hu CQ, et al. The type III secretion system of Vibrio alginolyticus induces rapid apoptosis, cell rounding and osmotic lysis of fish cells[J] . Microbiology, 2010, 156（9）：2864-2872.
[9] Burdette DL, Seemann J, Orth K. Vibrio VopQ induces PI3-kinase-independent autophagy and antagonizes phagocytosis[J] . Molecular Microbiology, 2009, 73（4）：639-649.
[10] Evans LD, Hughes C. Selective binding of virulence type III export chaperones by FliJ escort orthologues InvI and YscO[J] . FEMS Microbiology Letters, 2009, 293（2）：292-297.
[11] Cai SH, Lu YS, Wu ZH, et al. Loop-mediated isothermal amplification method for rapid detection of Vibrio alginolyticus, the causative agent of vibriosis in mariculture fish[J] . Letters in Applied Microbiology, 2010, 50（5）：480-485.
[12] Schwede T, Kopp J, Guex N, et al. SWISS-MODEL：an automated protein homology- modeling server[J] . Nucleic Acids Research, 2003, 31（13）：3381-3385.
[13] Xia Z, Chen W, Chang C, et al. A novel network and sparsity constraint regression model for functional module identification in genomic data analysis[J] . International Journal of Data Mining and Bioinformatics, 2013, 8（3）：311-325.
[14] Szklarczyk D, Franceschini A, Kuhn M, et al. The STRING database in 2011：functional interaction networks of proteins, globally integrated and scored[J] . Nucleic Acids Research, 2011, 39（Database issue）：561-568.
[15] Ghosh P. Process of protein transport by the type III secretion system[J] . Microbiology and Molecular Biology Reviews, 2004, 68（4）：771-795.
[16] Francis M S. Type III secretion chaperones：a molecular toolkit for all occasions[J] . Handbook of Molecular Chaperones：Roles, Structures and Mechanisms, 2010, 79-148.
[17] Li W, Liu Y, Sheng X, et al. Structure and mechanism of a type III secretion protease, NleC[J] . Acta Crystallographica Section D, 2014, 70（1）：40-47.
[18] Evans LD, Stafford GP, Ahmed S, et al. An escort mechanism for cycling of export chaperones during flagellum assembly[J] . Pro-ceedings of the National Academy of Sciences, 2006, 103（46）：17474-17479.
[19] Ibuki T, Uchida Y, Hironaka Y, et al. Interaction between FliJ and FlhA, components of the bacterial flagellar type III export apparatus[J] . Journal of Bacteriology, 2013, 195（3）：466-473.
[20] Neyt C, Cornelis GR. Insertion of a Yop translocation pore into the macrophage plasma membrane by Yersinia enterocolitica：requirement for translocators YopB and YopD, but not LcrG[J] . Molecular Microbiology, 2002, 33（5）：971-981.
[21] Turbyfill KR, Hartman AB, Oaks EV. Isolation and characterization of a Shigella flexneri invasin complex subunit vaccine[J] . Infection and Immunity, 2000, 68（12）：6624-6632.
[22] Cherradi Y, Hachani A, Allaoui A. Spa13 of Shigella flexneri has a dual role：chaperone escort and export gate-activator switch of the type III secretion system[J] . Microbiology, 2014, 160（1）：130-141.
[23] Diepold A, Wiesand U, Amstutz M, et al. Assembly of the Yersinia injectisome：the missing pieces[J] . Molecular Microbiology, 2012, 85（5）：878-892.
[24] Zhou Z, Pang H, Ding Y, et al. VscO, a putative T3SS chaperone escort of Vibrio alginolyticus, contributes to virulence in fish and is a target for vaccine development[J] . Fish and Shellfish Immunology, 2013, 35（5）：1523-1531.