摘要
自行构建的pMD18T-OmpU重组克隆质粒经BamHI/EcoRI双酶切后,定向插入原核表达质粒pGEX-4T-1中构建重组表达质粒pGEX-4T-1-OmpU。将重组表达质粒转化至大肠杆菌E.coliBL21进行IPTG诱导表达。表达产物经SDS-PAGE电泳及Western-blot分析显示,以包涵体形式表达的GST-OmpU融合蛋白的分子量约为62.9kD,可与鼠抗拟态弧菌外膜蛋白(Omps)免疫血清发生特异性反应,提示重组OmpU保留了天然Omps的抗原性。用纯化的融合蛋白免疫昆明系小鼠,以50LD50拟态弧菌(5×108CFU/mL)攻击,小鼠的免疫保护率为60%(9/15)。表明OmpU是拟态弧菌的保护性抗原之一,具有研发成为外膜蛋白基因工程亚单位苗的潜在价值。
Ascitic fluid disease ofEriocheir sinensis and fish caused by Vibrio mimicus is one of the most common epidemic diseases in Anhui province. Exploiting outer membrane protein vaccine against V.mimicus is one of the most effective means to prevent the disease. At present the domestic and foreign scholars have expounded that the outer membrane protein U （OmpU） of Vibrio cholerae is a protective antigen. Our recent studies indicate that four isolate strains of V.mimicus in Anhui all carry OmpU gene, and homology of the OmpU gene is high between V.mimicus and V. cholerae.To construct the prokaryotic expression plasmid for OmpU of V. mimicus in this study, the OmpU gene was inserted between BamHI and EcoRI sites of pGEX-4T-1 after cleavage by corresponding enzymes. The recombinant expression plasmid was named as pGEX-4T-OmpU. The fusion protein GST-OmpU with molecular weight of 62.9 kD by SDS-PAGE was expressed in E. coli BL21 at 37 ℃ after 1.0 mmol/L IPTG induction for 4 hours. Western blot analysis showed that mouse anti-Omps serum could specifically react to the recombinant OmpU, which suggested the recombinant OmpU retained antigenicity of natural Omps of V.mimicus. In order to determine the immunoprotection of fusion protein, experimental mice were immunized by purified fusion protein GST-OmpU, one positive （immunized with outer membranz protein of V.mimicus） and twe negative controls [immunized with glutathione s-transferase （GST） and non-immunized] were set. After three times of vaccination, mice were all challenged by V. mimicus isolate strain HX4 with 50% lethal dosage which was about 5×10^8 CFU/mL to mice. The mice immunized with Omps all survived.The mice immunized with GST-OmpU beared protective rates of 60%. The mice immunized with GST or non-immunized mice all died. All these results indicated that OmpU of V.mimicus may be one of the important protective antigens to V.mimicus for animals and it may play an important role in developing genetic engineering subunit vaccine of outer membrane protein to prevent ascitic fluid disease. [Journal of Fishery Sciences of China, 2008, 15 （2） ： 307-312]
出处
《中国水产科学》
CAS
CSCD
北大核心
2008年第2期307-312,共6页
Journal of Fishery Sciences of China
基金
国家自然科学基金项目(30771654)
安徽省自然科学基金项目(070411019)
安徽省教育厅自然科学基金项目(KJ2007A100)
