猪源支气管败血波氏杆菌百日咳杆菌黏附素基因的原核表达及其抗体检测ELISA方法

Expression of prn gene of Bordetella bronchiseptica and development of a re-combinant protein-based indirect ELISA for antibodies detection

摘要:

【目的】以猪源支气管败血波氏杆菌(Bordetella bronchiseptica, Bb)百日咳杆菌黏附素(PRN)基因的原核表达产物为抗原建立检测PRN抗体的间接ELISA方法。【方法和结果】利用谷胱甘肽-S-转移酶(GST)表达系统对PRN基因在大肠杆菌中进行融合表达。SDS-PAGE和Western blot 检测证实该基因获得高效表达, 产物易于纯化且具有良好的免疫学活性。通过凝血酶酶切GST-PRN并回收, 获得不含GST载体蛋白的PRN蛋白片段。以PRN蛋白片段为抗原建立检测天然PRN抗体的间接ELISA方法。该方法对猪巴氏杆菌病等7种常见细菌性疾病阳性血清的检测结果均为阴性, 其敏感性比乳胶凝集试验提高4～128倍, 能检测到人工感染14 d后的仔猪血清抗体IgG, 对临床送检的1,229份猪血清的检测阳性率为32.7%。ELISA方法对阳性猪场的监测结果预示了保育期仔猪的合群导致猪群大量感染支气管败血波氏杆菌。【结论】该方法具有特异性强、敏感性高、重复性好的特点, 可用于猪群PRN抗体水平监测和猪波氏菌病流行病学调查。

Abstract:

[Objective] We developed an indirect ELISA method for detecting Bordetella bronchiseptica (Bb) pertactin antibodies based on the recombinant pertactin protein expressed in Escherichia coli (DE3) strain. [Methods and results] The prn gene encoding Bb pertactin was fused to the downstream of glutathione S-transferase (GST) of pGEX-KG expression vector, resulting in the fusion expression plasmid pGEX-prn. SDS-PAGE showed that the GST-PRN fusion protein was expressed in high level in BL21 carrying pGEX-prn. The strong reactivity of the GST-PRN fusion protein, specifically with antiserum against porcine Bordetellosis caused by Bb HH0809, was identified by Western blot. The recombinant protein fragment of rPRN was purified from the GST-PRN fusion protein digested by protease thrombin with the purity of 93.1℅. The rPRN-based indirect ELISA was developed for detecting antibodies against PRN. The ELISA could detect positive samples in experimentally infected pigs fourteen days post inoculation and the degree of sensitivity was over 4 times higher than the latex agglutination test with the coating antigen of killed Bb. Thirty-two point seven percent of positive samples were detected in 1,229 clinical samples while no false positive results were found in detecting 7 antisera against porcine bacterial diseases. Sera samples from two bordetellosis-positive pig fields were tested by the indirect ELISA method and the results indicated that pigs were infected by Bb during the nursery periods. [Conclusion] The assay showed excellent specificity, sensitivity and reduplication, and can be useful for epidemiological survey and clinical diagnosis of swine bordetellosis.