溶藻弧菌转运蛋白TolB的免疫原性与免疫保护性

doi:10.13560/j.cnki.biotech.bull.1985.2015.07.021

生物技术通报 ›› 2015, Vol. 31 ›› Issue (7): 143-149.doi: 10.13560/j.cnki.biotech.bull.1985.2015.07.021

溶藻弧菌转运蛋白TolB的免疫原性与免疫保护性

庞欢瑛^1，2 周泽军^1，2 张燕飞^1，2 李静^1，2 邱明生^1，2 丁燏^1，2 简纪常^1，2 吴灶和^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广东省教育厅水产经济动物病害控制重点实验室，湛江 524088；3.仲恺农业工程学院，广州 510225)

收稿日期:2015-01-09 出版日期:2015-07-16 发布日期:2015-07-16
作者简介:庞欢瑛，女，博士，讲师，研究方向：水产经济动物病害控制；E-mail：phying1218 @163.com
基金资助:
国家自然科学基金项目(31402344)，广东省自然科学基金项目(S2013040014562)

Immunogenicity and Immunoprotection of Translocation Protein B(TolB)in Vibrio alginolyticus

Pang Huanying^1，2, Zhou Zejun^1，2, Zhang Yanfei^1，2, Li Jing^1，2, Qiu Mingsheng^1，2, Ding Yu^1，2, Jian Jichang^1，2, Wu Zaohe^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，Key Laboratory of Diseases Controlling for Aquatic Economic Animals of Guangdong Higher Education Institutions，Zhanjiang 524088；3. Zhongkai University of Agriculture and Engineering，Guangzhou 510225)

Received:2015-01-09 Published:2015-07-16 Online:2015-07-16

摘要/Abstract

摘要：

旨在研究溶藻弧菌(Vibrio alginolyticus)HY9901转运蛋白TolB作为疫苗候选抗原的可能性，根据已发表的全基因组序列，设计特异性引物PCR扩增tolB的基因全长序列。序列分析显示，该基因(GenBank登录号JQ846501)全长1 353 bp，共编码450个氨基酸残基。BLAST分析发现，溶藻弧菌tolB基因与其他已知弧菌的tolB具有较高的同源性，序列保守，可作为共同抗原候选蛋白。用原核表达的tolB蛋白免疫SPF级小鼠，制备多克隆抗体，ELISA效价达1∶40 000。免疫印迹表明鼠抗tolB血清能与诱导后的重组蛋白发生特异反应。为进一步研究tolB对石斑鱼(Epinephelus awoara)的免疫保护性，用TolB蛋白两次免疫石斑鱼，ELISA 检测发现，免疫石斑鱼血清的抗体效价在第4周达到峰值(1∶2 048)。攻毒实验结果表明TolB对石斑鱼的免疫保护率为76%。结果表明，溶藻弧菌转运蛋白TolB具有较好的免疫原性和免疫保护性，可作为弧菌亚单位疫苗的候选抗原。

关键词: 溶藻弧菌, tolB基因, 免疫原性, 免疫保护

Abstract:

To investigate the possibility of translocation protein B(TolB)from Vibrio alginolyticus HY9901 as a candidate antigen for vaccine production, the full length tolB gene was amplified by PCR and designed specific primers according to the whole genome sequence of V. alginolyticus published in GenBank. Sequence analysis revealed that tolB gene was 1 353 bp and encoded a putative protein of 450 amino acids(GenBank accession number：JQ846501). BLAST analysis showed that the tolB of V. alginolyticus had high genetic relationship with other known Vibrio, its sequence was conservative, and might be a common antigen candidate protein. The TolB protein of prokaryotic expression was injected into SPF mice to prepare polyclonal antibody, and its titer of ELISA reached 1∶40 000. Western blot indicated that TolB serum reacted specifically with the induced recombinant protein. Further the TolB protein was used as an antigen to immunize grouper(Epinephelus awoara)twice, and ELISA detection found that the antibody titer of the immunized grouper increased to the highest(up to 1∶2 048)in the fourth week. Challenge assay test showed that the immunoprotection rate of TolB to E. awoara was 76%. Therefore the TolB of V. alginolyticus has solid immunogenicity and immunoprotection, and it can be used as a candidate antigen for vaccine of Vibrio subunits.

Key words: Vibrio alginolyticus, tolB gene, immunogenicity, immunoprotection

庞欢瑛, 周泽军, 张燕飞, 李静, 邱明生, 丁燏, 简纪常, 吴灶和. 溶藻弧菌转运蛋白TolB的免疫原性与免疫保护性[J]. 生物技术通报, 2015, 31(7): 143-149.

Pang Huanying, Zhou Zejun, Zhang Yanfei, Li Jing, Qiu Mingsheng, Ding Yu, Jian Jichang, Wu Zaohe. Immunogenicity and Immunoprotection of Translocation Protein B(TolB)in Vibrio alginolyticus[J]. Biotechnology Bulletin, 2015, 31(7): 143-149.

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 Microbiol(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]. Annales de Dermatologie et de Venereologie, 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]黄志坚, 何建国. 溶藻弧菌外膜蛋白(Va-OMP)的免疫原性及免疫保护性[J]. 水产学报, 2006, 30(4)：538-543.
[7]潘燕华, 马悦, 赵东玲, 等. 鳗弧菌减毒活疫苗对牙鲆免疫效果的研究[J]. 食品与药品, 2009, 11(3)：12-15.
[8]曹剑香. 溶藻弧菌(Vibrio alginolyticus)疫苗及其外膜蛋白对南美白对虾(Penaeus vannamei)免疫功能的影响[D]. 湛江：湛江海洋大学, 2004.
[9]简纪常, 叶剑敏, 吴灶和. 溶藻弧菌脂多糖对石斑鱼免疫功能的影响[J]. 水生生物学报, 2004, 28(1)：103-105.
[10]梁海鹰, 夏立群, 吴灶和, 等. 溶藻弧菌HY9901鞭毛蛋白flaB基因的克隆及原核表达[J]. 水产学报, 2010, 34(1)：139-146.
[11]Liang H, Wu ZH, Jian JC, et al. Protection of red snapper(Lutjanus sanguineus)against Vibrio alginolyticus with a DNA vaccine containing flagellin flaA gene[J]. Letters in Applied Microbiology, 2011, 52(2)：156-161.
[12]Sturgis J. Organisation and evolution of the tol-pal gene cluster[J]. Journal of Molecular Microbiology and Biotechnology, 2001, 3(1)：113-122.
[13] Lloubès R, Cascales E, Walburger A, et al. The Tol-Pal proteins of the Escherichia coli cell envelope：an energized system required for outer membrane integrity?[J]. Research in Microbiology, 2001, 152(6)：523-529.
[14]Carr S, Penfold C, Bamford V, et al. The structure of TolB, an essential component of the tol-dependent translocation system, and its protein-protein interaction with the translocation domain of colicin E9[J]. Structure, 2000, 8(1)：57-66.
[15] Heilpern A, Waldor M. CTXφ infection of Vibrio cholerae requires the tolQRA gene products[J]. Journal of Bacteriology, 2000, 182(6)：1739-1747.
[16]Prouty AM, Van Velkinburgh JC, Gunn JS. Salmonella enterica serovar Typhimurium resistance to bile：identification and characterization of the tolQRA cluster[J]. Journal of Bacteriology, 2002, 184(5)：1270-1276.
[17]周泽军, 庞欢瑛, 丁燏, 等. 溶藻弧菌HY9901转运蛋白TolB的原核表达及条件优化和纯化[J]. 中国农学通报, 2013, 29(11)：55-59.
[18]Cai SH, Wu ZH, Jian JC, et al. Cloning and expression of the gene encoding an extracellular alkaline serine protease from Vibrio alginolyticus strain HY9901, the causative agent of vibriosis in Lutjanus erythopterus(Bloch)[J]. Journal of Fish Diseases, 2007, 30(8)：493-500.
[19]Godlewska R, Wi?niewska K, Pietras Z, et al. Peptidoglycan-associated lipoprotein(Pal)of Gram-negative bacteria：function, structure, role in pathogenesis and potential application in immunoprophylaxis[J]. FEMS Microbiology Letters, 2009, 298(1)：1-11.
[20]Paterson GK, Northen H, Cone DB, et al. Deletion of tolA in Salmonella Typhimurium generates an attenuated strain with vaccine potential[J]. Microbiology, 2009, 155(Pt 1)：220-228.
[21]Gerding MA, Ogata Y, Pecora ND, et al. The trans-envelope Tol-Pal complex is part of the cell division machinery and required for proper outer-membrane invagination during cell constriction in E. coli[J]. Molecular Microbiology, 2007, 63(4)：1008-1025.
[22]黄娇娇. 福氏志贺菌多药耐药相关基因的表达与耐药性关系研究[D]. 北京：中国农业科学院, 2010.
[23]吴灶和, 曹剑香, 简纪常, 等. 溶藻弧菌外膜蛋白对凡纳滨对虾免疫功能的影响[J]. 热带海洋学报, 2005, 24(6)：1-5.
[24]毛芝娟. 副溶血弧菌Vibrio parahaemolyticus主要外膜蛋白的克隆, 表达和免疫原性研究[D]. 杭州：浙江大学, 2007.
[25]张崇文, 于涟, 毛芝娟, 等. 哈维氏弧菌外膜蛋白OmpK基因的克隆及原核表达[J]. 水产学报, 2006, 30(1)：9-14.
[26]Li N, Yang Z, Bai J, et al. A shared antigen among Vibrio species：outer membrane protein-OmpK as a versatile Vibriosis vaccine candidate in Orange-spotted grouper(Epinephelus coioides)[J]. Fish and Shellfish Immunology, 2010, 28(5-6)：952-956.
[27]王妍妍, 李贵阳, 李杰, 等. 迟缓爱德华氏菌OppA蛋白的免疫原性及免疫保护分析[J]. 海洋科学, 2011, 35(5)：19-23.
[28]闫茂仓, 单乐州, 陈少波, 等. 三种创伤弧菌免疫原的制备及其对黄姑鱼的免疫保护效果[J]. 动物学研究, 2012, 33(5)：503-509.

溶藻弧菌转运蛋白TolB的免疫原性与免疫保护性

Immunogenicity and Immunoprotection of Translocation Protein B(TolB)in Vibrio alginolyticus

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