间接ELISA检测马链球菌兽疫亚种类M蛋白抗体方法的建立及应用.docVIP

  间接 ELISA 检测马链球菌兽疫亚种类 M 蛋 白抗体方法的建立及应用# 周红，范红结** 5 10 15 （南京农业大学，动物医学院，南京 210095） 摘要：根据马链球菌兽疫亚种 ATCC35246 类 M 蛋白（SzP）的基因序列，设计并合成引物， 以 ATCC35246 的基因组为模板，扩增出 1074bp 的 szp 基因片段。经 IPTG 诱导，目的基因 在大肠杆菌 BL21(DE3)中表达。SDS鉴定结果表明，获得了 54 kDa 的可溶性蛋白， 与目标蛋白一致。Western-blot 结果显示，表达的蛋白与猪源 ATCC35246 株康复血清反应 性良好。以纯化的 SzP 作为包被抗原，建立了 SEZ 血清抗体间接 ELISA 检测方法。对抗原 包被浓度、血清稀释度、封闭液的选择等反应条件进行了优化，结果表明，重组 SzP 的最佳 包被浓度为 0.625 μg/mL，标准阳性血清的最佳稀释倍数为 1:40，最适封闭液为含 0.5%脱 脂乳的 PBS 溶液，待检血清的最佳孵育时间为 60 min，酶标二抗的最佳作用时间为 45 min。 用本试验所建立的 SEZ 血清抗体间接 ELISA 检测方法，对采自河南、山东、安徽、上海、 江苏等地猪场的 1080 份血清进行检测。结果表明，上述 5 地猪场血清样本 SEZ 抗体的阳性 率分别为 33.3%、16.5%、21.8%、13.9%和 13.5%。综上，本试验所建立的间接 ELISA 检测 方法的特异性和重复性良好，具有重要的临床应用价值。 关键词：马链球菌兽疫亚种；类 M 蛋白；间接 ELISA 中图分类号：S855.1 20 Development of an indirect ELISA for the detection of antibodies against Streptococcus equi ssp. zooepidemicus ZHOU Hong, FAN Hongjie (Nanjing Agricultrual University, College of Veterinary Medicine, Nanjing 210095) 25 30 35 40 Abstract: A 1074bp fragment of M-like protein(SzP) was amplified by PCR according to the gene sequences ofStreptococcus equi ssp. Zooepidemicus(SEZ)ATCC35246 strain. After being induced by IPTG, the gene szp was expressed in E. coli BL21 (DE3). The result of SDSshowed that a 54 kDasoluble protein was expressed, which was consistent with the target protein. Western-blot analysis showed that the expressed protein had strong reactogenicitywith the rehabilitated swine serum.In the optimized indirect ELISA, the recombinant protein was coated at 0.625 μg/mL.The optimal dilution of standard positive serum was 1:40. The optimal blocking buffer was 0.5% skim milk in PBS. The optimalincubation time of tested serum was 60 min. The optimalreaction time of HRP-SPA was 45 min. 1080 serum samples from Henan, Shandong, Anhui, Shanghai and Jiangsuprovince were tested for the SEZ serum antibodiesby this indirect ELISA method. The results showed that the positive rates of these 5 provinces were33.3%, 16.5%, 21.8%, 13.9% and 13.5%, respectively. In summar