嗜水气单胞菌粘附素和外膜蛋白A基因克隆与结构预测

Cloning and Structure Prediction of Adhesion Gene and Outer Membrane Protein A Gene from Aeromonas hydrophila

采用PCR方法扩增嗜水气单胞菌（Aeromonas hydrophila）外膜蛋白相关基因,即粘附素（Aha）、外膜蛋白A（OmpA）基因全长序列,进行DNA测序及生物信息学分析。结果表明：Aha基因长1 314 bp,推导编码355 aa,5～24 aa区域为跨膜螺旋,26～355 aa区域为革兰氏阴性菌孔蛋白结构域（PF00267）,Aha蛋白三级结构与模板（PDB：1PHO_A）相似性达28.21%;OmpA基因长1 102 bp,推导编码338 aa,含有OmpA跨膜结构域（PF01389）和OmpA结构域（PF00691）,预测OmpA蛋白抗原表位位于64～69 aa、160～171 aa、244～249 aa、259～274 aa和295～301 aa区域,OmpA蛋白三级结构OmpA跨膜结构域和OmpA结构域与模板（PDB：1BXW_A,PDB：4ERH_A）相似性分别为27.45%和57.58%;进一步采用拉马钱德兰图（Ramachandran plot）检测分析,与Aha、OmpA基因蛋白三级结构预测结果一致。本研究有助于理解嗜水气单胞菌致病的分子机制,为鱼类疾病防治及疫苗应用提供科学参考。

Two outer membrane protein-related genes from the Zfl strain of A eromonas hydrophila, including adhesion （Aha） gene and outer membrane protein A （OmpA） gene, were amplified by PCR and the structure of them were then analyzed by bioinformatics methods. The results showed that the A ha gene sequence was 1 314 bp long, encoding a protein of 355 amino acids which has a transmembrane helix position （5-24） and gram-negative porin domain （PF00267）; the OmpA gene sequence was 1 102 bp long, encoding a protein of 338 amino acids which has OmpA-like transmembrane domain （PF01389） and OmpA domain （PF00691）. Moreover, the antigenic epitopes of OmpA protein were predicted to be located in the five regions （ie： 64-69, 160-171,244-249, 259-274 and 295-301）. Homology modeling was used to construct 3D structure of Aha protein with the standard model （PDB： 1PHO_A） and OmpA protein with the known crystal 3D structure （PDB： 1BXW_A ＆ PDB： 4ERH_A）, respectively. In conclusion, these results help to understand the molecular mechanisms of A eromonas hydrophila pathogenesis, which will provide a scientific reference for the prevention and treatment offish diseases and vaccine applications.