摘要： 为构建水产致病菌溶藻弧菌附着定植因子ACFA原核表达载体,优化ACFA蛋白的诱导表达条件,制备ACFA蛋白小鼠多克隆抗体。通过分子克隆获得ACFA蛋白的表达菌株,利用切胶纯化获得ACFA蛋白,免疫小鼠制备多克隆抗体,采用ELISA法检测抗体滴度为1:3 200倍,Western-Blotting法检测抗血清特异性较好。通过正交试验, 获得ACFA菌株的最佳表达条件为:诱导时菌液OD₆₀₀值0.5,加IPTG终浓度0.3 mmol/L,诱导时间8 h,诱导温度32 ℃; 最佳培养条件为:葡萄糖浓度0,转速230 r/min,装液量50 mL。

关键词: 溶藻弧菌, ACFA蛋白, 正交试验, 多克隆抗体

Abstract: To construct prokaryotic expression vector of aquatic pathogenic Vibrio alginolyticus accessory colonization factor A (ACFA), optimize the expression condition of ACFA and prepare the polyclonal antibodies of mouse anti-ACFA.The ACFA expression strain was obtained by molecular clone.ACFA was purified by the way of the gel slices, immunized mice and prepared the polyclonal antibody.The antibody titer was 1:3 200 by ELISA, and Western-Blotting proved that the antiserum had good specificity.By the method of orthogonal experiment, the optimal inducing expressing condition of ACFA protein:strain OD₆₀₀ value, IPTG final concentration, inducing time and temperature, which were 0.5, 0.3 mmol/L, 8 h, 32 ℃, respectively;the optimal culture condition:glucose concentration, rotation rate, and medium volume, which were 0, 230 r/min and 50 mL, respectively.

Key words: Vibrio alginolyticus, ACFA protein, Orthogonal experiment, Polyclonal antibody

中图分类号: 

• Q816