鳜鱼嗜水气单胞菌GYK1株外膜蛋白A基因的克隆及其生物信息学分析  

Cloning and bioinformatics of outer membrane protein A gene of Aeromonas hydrophila GYK1 strain

作　　者：付小哲[1] 方翔[1] 李宁求[1] 林强[1] 潘厚军[1] 石存斌[1] 黄志斌[1] 吴淑勤[1] 

机构地区：[1]中国水产科学研究院珠江水产研究所农业部渔用药物创制重点实验室广东省水产动物免疫技术重点实验室,广东广州510380

出　　处：《中国生物制品学杂志》2013年第9期1222-1227,共6页Chinese Journal of Biologicals

基　　金：国家科技支撑计划(2006BAD03B05);国家自然科学基金(31202032);广东省自然科学基金(7004728)

摘　　要：目的克隆鳜鱼嗜水气单胞菌GYK1株外膜蛋白A(outer membrane protein A,OmpA)基因,并进行生物信息学分析。方法根据已发表的细菌ompA序列,通过比对保守区域(88~107 bp,988~1 007 bp)设计简并引物,通过PCR技术从鳜鱼嗜水气单胞菌GYK1株扩增ompA基因核心序列,采用热不对称交错PCR(thermal asymmetric interlaced PCR,TAIL-PCR)法扩增ompA基因全长及其上下游序列;应用Vector NTI 9.0对ompA序列进行拼接,应用Vector NTI 9.0、SignalP 3.0对OmpA蛋白的基本参数、信号肽等进行预测分析;通过Blastn分析嗜水气单胞菌GYK1株ompA基因序列与其他菌种ompA序列的同源性,并构建系统进化树及对其蛋白的三维结构进行分析。结果ompA基因全长为2 004 bp,其中包括ORF 1 059 bp,编码352个氨基酸,5′端侧翼序列271 bp,3′端侧翼序列574 bp;OmpA蛋白相对分子质量约37 900,等电点为4.94,OmpA蛋白序列N-末端的前20个氨基酸残基为信号肽序列;根据22种细菌的OmpA蛋白序列构建的系统进化树显示,嗜水气单胞菌GYK1株与嗜水气单胞菌SSU株的亲源关系最近,相似性达95.5%;三维结构分析表明,OmpA蛋白N-末端功能区是由8个反向平行的β-折叠片构成的β-折叠桶;OmpA蛋白Asn25~Ala205片段4个环区的平均柔性显著高于其余部位的柔性。结论成功克隆了鳜鱼嗜水气单胞菌GYK1株ompA基因,并对OmpA蛋白的相关生物信息学进行了分析,为嗜水气单胞菌呈递外源蛋白基因工程疫苗的研究奠定了基础。Objective To clone the outer membrane protein A （OmpA） gene of Aeromonas hydrophila GYK1 strain and analyze its bioinformatics. Methods According to the sequence of published ompA gene, a pair of degenerate primers were designed to amplify the core sequence of ompA gene of GYK1 strain by PCR, based on which thermal asymmetric interlaced PCR （TAIL-PCR） was performed to amplify further flanking regions of the gene. The amplified ompA gene fragments were spliced by using Vector NTI 9. 0 software, predicted for basic parameters and signal peptide of OmpA by Vector NTI 9. 0 and SignalP 3. 0 software, and analyzed for homologies to the ompA gene of other bacterial strains by BlastN software, based on which a phylogenetic tree was constructed, and the three-dimensional structure of OmpA was analyzed. Results The cloned ompA gene of GYK1 strain, at a full-length of 2 004 bp, consisting of an ORF at a length of 1 059 bp encoding 352 amino acids, a 5＇-terminal flanking sequence at a length of 271 bp and a and 3＇-terminal flanking sequence at a length of 574 bp. The relative molecular mass and isoeleetric point of OmpA were about 37 900 and 4. 94, respectively, while the signal peptide consisted of amino acid residues 1 -20 at N-terminus. The phylogenetic tree constructed based on the OmpA sequences of 22 bacterial strains revealed highest homology（95. 5%） of GYK1 strain to SSU strain of A. hydrophila. Three-dimensional structure model showed that the N-terminal domain of OmpA was - barrel consisting 8 anti-parallel ^-sheets, while the average flexibility of four loop regions in segment Asn25 - Ala205 were significantly higher than those of other regions. Conclusion The ompA gene of A. hydrophila GYK1 strain was successfully cloned, of which the bioinformatics was analyzed. It laid a foundation of development of recombinant vaccine based on A. hydrophila-delivered exogenous proteins.

关 键 词：嗜水气单胞菌 外膜蛋白A 基因克隆 生物信息学 

分 类 号：S941.42[农业科学—水产养殖] Q785[农业科学—水产科学]