羊布鲁菌BCSP31蛋白表达纯化及其单克隆抗体的制备

Expression and purification of BCSP31 protein of Brucella melitensis and pre-paration of monoclonal antibodies against BCSP31

目的:表达并纯化羊布鲁菌BCSP31蛋白,制备其单克隆抗体(mAb).方法:采用GST亲和层析纯化的方法纯化BCSP31蛋白.用纯化的BCSP31蛋白免疫小鼠,将小鼠脾细胞与骨髓瘤Sp2/0细胞融合,ELISA间接法筛选阳性克隆,3次克隆化后建立杂交瘤细胞系.采用小鼠腹腔接种杂交瘤细胞制备腹水,正辛酸-硫酸铵法纯化mAb.采用Western Blot和ELISA等方法鉴定mAb特性.结果:GST-BCSP31融合蛋白在25℃诱导时为可溶性表达,经亲和层析纯化,获得BCSP31纯化蛋白0.5 g/L;Western Blot检测结果显示所获蛋白可与兔抗布鲁菌阳性血清特异性反应.获得2株mAb,分别命名为1F1,1E5.结论:BCSP31蛋白的纯化及其mAb的制备为布鲁菌病的诊断与防治奠定了一定的物质基础.

AIM： To express and purify BCSP31 protein from Brucella melitensis and prepare monoclonal antibodies （mAb） against it. METHODS： The mAb against BCSP31 was made by routine procedures： purifying protein via GST affinity chromatography, immunizing mice with purified recombinant protein, fusing cells, cloning by indirect ELISA, preparing ascites and purifying ascites by caprylic acid and ammonium sulfate. The characteristics of the mAb were analyzed by Western Blot and ELISA. RESULTS： The fusion protein GST-BCSP31 was expressed in soluble form at 25 ℃. The purified BCSP31 protein 0. 5 g/L was obtained and was reactive with the positive serum from Brucella melitensis immunized rabbits. Two mAb strains, named 1F1 and 1E5, were obtained. CONCLUSION： Purified BCSP31 protein and its mAb may play some important roles in the diagnosis and prevention of Brucellosis.