溶藻弧菌外膜蛋白OmpU的克隆、表达条件优化及多克隆抗体制备

Cloning,Optimization Expression Conditions and Polyclonal Antibody Preparation of Vibrio alginolyticus Outer Membrane Protein OmpU

Omp U蛋白为溶藻弧菌主要外膜蛋白,具有很好的免疫原性,在疫苗上有很好的应用前景。本试验通过分子克隆获得Omp U蛋白的表达菌株,利用切胶纯化获得Omp U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∶3200倍,WesternBlotting法检测抗血清特异性较好。通过正交试验,获得Omp U菌株的最佳表达条件为:诱导时菌液OD600值1,加IPTG终浓度0.1mmol/L,诱导时间8 h,温度32℃;最佳培养条件为:葡萄糖浓度0.4%,转速230 r/min,装液量50 m L。为Omp U工业发酵、蛋白功能与疫苗开发研究奠定基础。

OmpU, a major outer membrane of Vibrio alginolyticus （ V. alginolyticus）, have good immunogenicity and a good application prospect in vaccine development. The OmpU expression strain was obtained by molecular clone. OmpU was purified by the way of the gel slices, immunized mice and prepared the polyclonal antibody. The antibody fiter was 1 ： 3200 by ELISA, and Western-blotting proved that the antiserum had good specificity. By the method of orthogonal experiment, the optimal inducing expressing condition of OmpU protein： strain ODe,00 value, IPTG final concentration, inducing time and temperature, which were 1,0.1 retool/L, 8 h, 32℃, respectively; the optimal culture condition： glucose concentration, rotation rate, and medium volume, which were 0.4 %, 230 r/min and 50 mL, respectively. These work laid the groundwork for the industrial fermentation, protein function and vaccine development of OmpU.