哈维氏弧菌硫氧还蛋白还原酶在大肠杆菌中的表达及对大菱鲆的免疫保护作用

Expression of thioredoxin reductase of Vibrio harveyi SF1 in bacterium Enterobacter coli and immunogenicity in turbot Scophthalmus maximus

从致病性哈维氏弧菌Vibrio harveyi SF1基因组中扩增获得含硫氧还蛋白还原酶(trxR)基因的1 133bp目的片段,连接至载体pMD19-T Simple。测序结果表明,目的片段含有960 bp trxR基因阅读框,编码为319个氨基酸残基的蛋白质。Blast分析结果显示,硫氧还蛋白还原酶蛋白氨基酸序列与哈维氏弧菌、溶藻胶弧菌、副溶血弧菌、灿烂弧菌、弗氏弧菌的硫氧还蛋白还原酶蛋白序列的相似性分别为99%、98%、96%、94%、92%。构建表达质粒pET-28a(+)/TrxR后转化至大肠杆菌E.coli BL21(DE3)中,用IPTG诱导表达,SDS-PAGE分析显示,重组蛋白的相对分子质量为34 000。用Ni琼脂糖亲和层析柱分离得到纯化的重组蛋白,将该蛋白以50μg/尾的剂量肌肉注射免疫大菱鲆Scophthalmus maximus,用ELISA法检测免疫1～4周内鱼血清中特异性抗体的效价。结果表明,特异性抗体效价持续升高,第2周则达到1∶128。免疫4周后用哈维氏弧菌SF1对大菱鲆进行人工感染,对照组鱼的死亡率为100%,免疫组鱼的死亡率为25%,相对免疫保护力为75%。试验表明,哈维氏弧菌TrxR蛋白具有较好的免疫原性,可作为潜在的亚单位疫苗用于哈维氏弧菌病的免疫预防。

A thioredoxin reductase（trxR） gene was cloned by PCR amplification from the chromosomal DNA of bacterium Vibrio harveyi SF1.The ORF of the TrxR gene was shown to consist of 960 base pairs,encoding a polypeptide of 319 amino acids.Sequence analysis showed that the amino acid sequence was found 99% similarity with thioredoxin reductases of Vibrio harveyi,98% with Vibrio alginolyticus,96% with Vibrio parahaemolyticus,94% with Vibrio splendidus and 92% with Vibrio fluvialis.A recombinant plasmid pET28a（＋）/TrxR was constructed by inserting the trxR gene into pET28a（＋）.The recombinant plasmid was further transferred into Enterobacter coli BL21（DE3） and expressed with IPTG induction.The protein was expressed and purified by Ni（＋）-affinity chromatography,and showed a molecular weight of 34 000 on SDS-PAGE.Turbot was immunized with 50 μg of the recombinant protein and the immunized fish was challenged with 0.1 mL of V.harveyi（3.9×108 CFU/mL） for four weeks.The protein vaccine were assessed by ELISA at different days post-vaccination.The antibody titer was found to be 1∶ 128.The fish in the immunized group had relative percentage survivals（RPS） of 75%.Significant specific antibody responses were detected in the vaccinated fish by indirect ELISA.