中文摘要：

副溶血弧菌是引起细菌性食物中毒的主要食源性致病菌,耐热直接溶血毒素（theromostable direct hemolysin,TDH）是其公认的主要致病因子。本研究根据已发表基因序列（Gen Bank登录号：M10069）设计引物,以副溶血弧菌SHJLA株基因组DNA为模板,扩增编码TDH蛋白的基因,克隆至表达质粒p ET28a（＋）中,重组质粒经限制性酶切鉴定后测序,结果表明插入片段长度为576 bp。重组原核表达质粒p ET28a-tdh转化至大肠杆菌BL21（DE3）中,经诱导可表达分子量约为27 k Da的融合蛋白,主要以包涵体形式表达。对包涵体进行复性,Western blot分析表明经过复性后的重组蛋白能够被感染副溶血弧菌SHJLA株的小鼠血清识别。重组蛋白免疫家兔,ELISA检测多抗血清效价在1：256 000以上。将该多抗初步应用于斑点-ELISA（DotELISA）方法的建立,TDH阳性的致病性副溶血弧菌检测到阳性斑点,而TDH阴性的副溶血弧菌及其他细菌未检测到特异性斑点。本研究可为深入研究TDH的蛋白功能,建立致病性副溶血弧菌免疫学快速诊断方法以及保护性疫苗的研制提供研究基础。

英文摘要：

Vibrio parahaemolyticus is a major food-borne pathogen causing bacterial food poisoning and theromostable direct hemolysin（TDH） is the principal pathogenic factor.In this study,primers were designed according to the TDH gene sequence of Vibrio parahaemolyticus published in GenBank（M10069）.With the specific primers and DNA from strain SHJLA,the tdh gene fragments were amplified in PCR and cloned into the pET28a（＋） vector.Then,the recombinant plasmid pET28-tdh was transformed into E.coli BL21（DE3） and induced with IPTG.The cloned gene was 576 bp in length.The target recombinant protein with the molecular weight of27 kDa was hyperexpressed in the form of inclusion body as analyzed in SDS-PAGE and Western blot.After being refolded and purified,the recombinant protein was used to immunize rabibits.The obtained polyclonal antibodies against TDH was titrated higher than 1：256 000 in ELISA.A dot-ELISA method based on polyclonal antibodies was preliminary established to detect pathogenic V.parahaemolyticus,and the specific spots only detectable in the TDH positive V.parahaemolyticus.The results from the present study will put forward the prospects for in-depth research on TDH function,development of a rapid immunodiagnostic method of the TDH toxin and utilization as a vaccine candidate against V.parahaemolyticus infection.