中文摘要：

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。

英文摘要：

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.