大黄鱼源杀香鱼假单胞菌外膜蛋白OmpA的原核表达及免疫原性分析

Prokaryotic Expression and Immunogenicity Analysis of Outer Membrane Protein A from Pseudomonas plecoglossicida,A Pathogen of Larimichthys crocea

外膜蛋白A(OmpA)是革兰氏阴性菌主要的外膜蛋白之一,在多种致病菌中表现出较强的免疫原性,是具有潜力的候选疫苗。为分析大黄鱼内脏白点病病原杀香鱼假单胞菌OmpA类外膜蛋白的免疫原性,本研究选取了杀香鱼假单胞菌基因组中存在的3个OmpA类重组蛋白(依次命名为P1,P2,P5,Genbank收录号分别为EPB94769.1,EPB94930.1,EPB95835.1),通过构建pET30a-OmpA表达载体、转化BL21诱导表达、His标签纯化等技术,获得3个纯化的OmpA类重组蛋白,分子量大小依次为25 kD(P1),32 kD(P2),35 kD(P5)。用制备的新西兰兔抗杀香鱼假单胞菌多克隆抗体,对获得的纯蛋白进行免疫原性Western Blot检测,结果表明:重组蛋白P1,P2,P5均能与兔抗发生特异结合,结合强度P5>P2>P1。本研究首次获得了杀香鱼假单胞菌OmpA重组蛋白并初步鉴定了其免疫原性,为杀香鱼假单胞菌的疫苗设计和筛选提供了有益参考。

Outer membrane protein A(OmpA) is one of the major outer membrane proteins of Gram-negative bacteria, exhibits strong immunogenicity in a variety of pathogens and is a potential candidate vaccine. To analyze the immunogenicity of the OmpA from Pseudomonas plecoglossicida, a pathogen caused the visceral granulomas syndrome in large yellow croaker, we picked 3 OmpA (named P1, P2, P5, with the GenBank Accession number of EPB94769.1, EPB94930.1, EPB95835.1 respectively) presented in the P. plecoglossicida genome. pET30a-OmpA expression vectors were constructed and transformed into BL21,followed by induced expression and His-Tag purification, we finally got 3 purified recombinant protein, with the molecular size of 25 kD(P1), 32 kD(P2), 35 kD(P5) respectively. To detect the immunogenicity of the purified proteins, a New Zealand rabbit anti-P. plecoglossicida polyclonal antibody was prepared and Western Blot test was carried, the result shows that: both the recombinant proteins P1, P2, P5 can specifically bind to the polyclonal antibody, but the binding strength is varied: P5>P2>P1. We here in this study for the first time obtained the recombinant OmpA of P. plecoglossicida and preliminarily evaluated their immunogenicity, thus provided useful reference for the design and screening of P. plecoglossicida vaccine.