【摘要】 目的 为了初步评价基于FimD、PilN和PilV蛋白优势表位设计的重组蛋白免疫保护效果。方法 将FimD、PilN和PilV蛋白的优势B细胞表位串联，构建原核表达载体，然后在BL21(DE3)大肠杆菌中进行原核表达及纯化；用弗氏佐剂与重组蛋白充分混匀后免疫小鼠，收集血清，利用间接ELISA方法检测抗体效价，并进行抗原性的检测；三免后进行攻毒保护实验并对小鼠肝脏、脑和脾脏载菌量进行测定。结果 重组蛋白主要以包涵体形式表达。Western-blot结果显示出现一条特异性条带，与预期的重组蛋白大小相符，表明成功制备出多表位抗原。重组蛋白免疫小鼠后血清特异性抗体效价高达1:204 800。试验组小鼠攻毒后存活率为66%,对照组小鼠肝脏、脑和脾脏载菌量均显著高于试验组。结论 重组蛋白具有良好的抗原性和一定的免疫保护作用，具有研发牛源致脑炎大肠杆菌多表位疫苗的潜在价值。更多还原

【Abstract】 Objective In order to preliminarily evaluate the immune protection effect of recombinant proteins designed based on the dominant epitopes of FimD, PilN and PilV proteins.Methods The dominant B cell epitopes of FimD, PilN and PilV proteins were concatenated to construct a prokaryotic expression vector, which was then expressed and purified in BL21(DE3) E. coli. Preparation of mixed multi-epitope vaccine of Freund’s adjuvant and recombinant protein. After immunizing mice, the serum was collected for antibody titer and antigenicity detection by indirect ELISA method. After three immunizations, the bacteria protection experiment was carried out, and the amount of bacteria in the liver, brain and spleen of mice was measured. Results The recombinant protein was mainly expressed as inclusion bodies, and the amount of expression was the highest at 8 h. Western-blot results showed that there had a specific band, which was consistent with the expected size of the recombinant protein, indicating that the multi-epitope antigen was successfully prepared. After the mice were immunized by recombinant protein, the serum specific antibody titer was as high as 1:204 800. The survival rate of mice in the experimental group was 66% and the amount of bacteria in the liver, brain and spleen of the mice in the control group was significantly higher than that of the experimental group. Conclusion The recombinant protein had good antigenicity and had a certain immune protection, and had the potential value of developing a bovine encephalitis-causing E. coli multi-epitope vaccine.更多还原