猪链球菌2型人源株MRP的表达及其单克隆抗体的制备与应用

将猪链球菌2型(SS2)人源株Habb mrp基因进行截短修饰后,克隆于pGEX4T-2载体中,转化大肠杆菌诱导表达约61 000的融合蛋白MRP-GST,该蛋白经凝血酶作用去除重组蛋白中的GST标签,得到约35 000纯化的MRP.Western blotting证实MRP可被SS2阳性血清特异性识别.以纯化的MRP抗原免疫BALB/c小鼠,取免疫鼠脾细胞与骨髓瘤细胞SP2/0融合,间接ELISA进行筛选获得了6株能稳定分泌抗MRP特异性单抗的细胞株.特异性试验表明该6株单抗与SS2的另外2种蛋白、大肠杆菌均不发生交叉反应.以2138单抗腹水和SS2多克隆抗血清建立夹心ELISA对71株标准菌株和122株猪链球菌野毒株进行MRP的表征鉴定,标准株检测结果与背景符合率为为97.2%(69/71),其中34株标准血清型菌株检测的符合率为100%.表明该方法可用于猪链球菌的快速诊断和流行病学调查.

Expression of human Streptococcus suis 2 muramidase released protein and preparation and application of its monoclonal antibodies

The mrp gene of SS2 human strain Habb was truncated and cloned into a prokaryotic expression vector pGEX4T-2,and a fusion-expressed protein MRP-GST of 61 000 was obtained in E.coli.The GST was cut from MRP-GST with thrombin protease to gain the purified MRP of 35 000 which showed a strong reaction to the SS2 positive sera in Western blotting.BALB/c mice were immunitied intraperitoneally with purified MRP protein.Murine myeloma cells were fused with the splenocytes of the immunized mice after the third immunization.An indirect ELISA coated with purified MRP was used to screen hybridomas for production of specific antibody.The six McAb recognized MRP specially.According to the results of 71 standard strains (48 SS and 34 SS2)by Sandwich ELISA,the coincidence rate was 97.2%,but for 34 SS standard serotype was 100 %.The ELISA method has a potential value for clinical and epidemiological applicationgs.