鳗鲡病原性气单胞菌外膜蛋白基因全长的克隆与抗原决定簇分析

生物技术通报 ›› 2013, Vol. 0 ›› Issue (7): 144-152.

鳗鲡病原性气单胞菌外膜蛋白基因全长的克隆与抗原决定簇分析

郭松林,王玉,关瑞章,冯建军,林鹏,杨求华

（集美大学水产学院鳗鲡现代产业技术教育部工程研究中心，厦门 361021）

收稿日期:2012-12-27 修回日期:2013-07-19 出版日期:2013-07-19 发布日期:2013-09-02
作者简介:郭松林，男，博士，副教授；E-mail: gsl@jmu.edu.cn
基金资助:
国家自然科学基金项目（31001136），福建省自然科学基金项目（2010J01214），福建省教育厅项目（JA10191），福建省教育厅资助省属高校科研专项（JK2011029）

Full-length Gene Cloning and Common Antigen Analysis of Outer Membrane Protein of Pathogenic Aeromonas Isolated from Diseased Eels

Guo Songlin, Wang Yu, Guan Ruizhang, Feng Jianjun, Lin Peng, Yang Qiuhua

（Fisheries College，Jimei University，Engineering Research Centre of Eel Modern Technical Industry，Ministry of Education，Xiamen 361021）

Received:2012-12-27 Revised:2013-07-19 Published:2013-07-19 Online:2013-09-02

摘要/Abstract

摘要： 为寻找存在于鳗鲡病原性气单胞菌间的共同外膜蛋白保护性抗原。通过NCBI/GenBank数据库检索，找出气单胞菌多种外膜蛋白的序列信息，经比对后找到一条高度保守的基因序列片段。据相应片段从嗜水气单胞菌（Aeromonas hydrophila）和杀鲑气单胞胞菌（A. salmonicida）的全基因组序列中钓出Ⅱ型孔蛋白基因的全长序列及该基因区域之外两端的序列。根据嗜水气单胞菌和杀鲑气单胞胞菌这两端序列的保守区域设计一对特异性引物，用该引物扩增到鳗鱼病原库30株气单胞菌中11株菌的Ⅱ型孔蛋白基因全长，经测序后获得10株菌的Ⅱ型孔蛋白基因全长序列。选择遗传距离居中菌株，对该基因推导的表达产物进行蛋白结构预测、亲水性和抗原决定簇分析后发现，这些蛋白均具有三级结构，亲水性强且具有丰富的抗原决定簇。不同菌株的多个抗原决定簇经比对分析后，寻找到存在于多株鳗鲡病原性气单胞菌间的6个保守抗原决定簇。为养殖鳗鲡病原菌外膜蛋白共同保护性抗原的基因工程疫苗研究奠定了理论基础。

关键词: 外膜蛋白, 病原性气单胞菌, 全长基因克隆, 共同抗原决定簇, 鳗鲡

Abstract: In order to search the common antigen of out membrane protein of pathogenic Aeromonas isolated from diseased eels, 17 Aeromonas gene sequences of outer membrane protein（OMP）from the NCBI/GenBank were compared and the conserved fragments of one OMP（porin II）were determined. According to the whole genome sequences of Aeromonas hydrophila and Aeromonas salmonicida, the full-length gene sequences of porin II（ompII）were ascertained and the related up and down fragments out of ompII sequences were ascertained. One pair of specific primers was designed at the up and down fragments. Using the primers, 11 OmpII genes were successfully amplified by temperate DNA of 30 strains of pathogenic Aeromonas, and 10 of 11 products were sequenced. Protein structure, hydrophilicity and epitope were analyzed using the predicted expression products of a full-length sequence of ompII. The results showed that the predicted protein was a trans-membrane protein with tertiary structure, and it was also a hydrophilic protein with multiple epitopes. The study provided theoretical basis on gene engineering vaccine designing through common antigens of OMP of pathogenic Aeromonas isolated from farming eels.

Key words: Outer membrane protein, Pathogenic, Aeromonas, Full-length gene cloning, Common epitopes, Eel

郭松林,王玉,关瑞章,冯建军,林鹏,杨求华. 鳗鲡病原性气单胞菌外膜蛋白基因全长的克隆与抗原决定簇分析[J]. 生物技术通报, 2013, 0(7): 144-152.

Guo Songlin, Wang Yu, Guan Ruizhang, Feng Jianjun, Lin Peng, Yang Qiuhua . Full-length Gene Cloning and Common Antigen Analysis of Outer Membrane Protein of Pathogenic Aeromonas Isolated from Diseased Eels[J]. Biotechnology Bulletin, 2013, 0(7): 144-152.

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