胸膜肺炎放线杆菌毒素apxIVA基因的克隆与表达及间接ELISA方法的建立

Cloning and Expression of the apxIVA Gene of Actionbacillus pleuroneumonia e and Development of an Indirect ApxIVA-ELISA

参照APP血清 1型apxIVA基因序列合成了一对特异性引物 ,从本实验室分离鉴定的胸膜肺炎放线杆菌 (APP)血清 2型中扩增了apxIVA基因 5′端 2 4 4 5bp的片段。将该片段克隆到原核表达载体pET_2 8b的T7启动子下游 ,与 6×HisTag融合 ,再转化大肠杆菌BL2 1(DE3) ,在IPTG的诱导下表达大小约 90kD的蛋白。表达产物以包涵体的形式存在 ,且能与APP标准阳性血清发生特异性反应。将包涵体变性和复性后包被酶标板建立了间接ELISA方法 (ApxIVA_ELISA) ,特异性良好。用ApxIVA_ELISA检测猪胸膜肺炎三价 (1、2和 7型 )灭活菌苗和基因工程类毒素菌苗免疫猪血清抗体均为阴性 ,而 1、2、7型APP活菌感染动物的血清抗体均为阳性。实验证明 ,ApxIVA_ELISA不仅可以用于检测所有血清型APP的抗体 ,而且还可以用于APP自然感染猪和灭活菌苗免疫猪的鉴别诊断。

ApxⅣ, a forth RTX toxin indentified in Actionbacillus pleuropneumoniae recen tly, is expressed by all A. pleuropneumoniae regardless the ser otypes and inducible only in vivo toxin, so it is the optimal t o develop species-specific and differentiated diagnostic assay. Here the 2445bp DNA fragment of apxIVA gene of A. pleuroneumoniae was amplified and fused in-frame to the downstream of the T7 promoter and 6 His Tag of the pr okaryotic expression vector pET-28b. The construct was transformed into E. col i BL21(DE3). After induction by 1.0 mol/L IPTG, a recombinant protein about 9 0kD in s ize, designed as ApxIVAN, was detected, which was present as inclusion bodies an d reacted specifically with swine antisera to the APP-serotype-1 by dot-blot. An indirect ELISA (ApxIVA-ELISA) was developed using purified recombinant ApxIVAN from the inclusion bodies as described previously, which had excellent specifici ty to A. pleuroneumoniae. Using the ApxIVA-ELISA, the ApxIV ant ibodies were not detected in the inactivated APP bacterins vaccinated pigs, but were detected in A. pleuropneumoniae serotype 1, 2 and 7 infect ed pigs and mice. These results suggested that ApxIVA-ELISA can be used not only to detect all serotypes of APP, but also to differentiate the naturally infecte d and inactivated vaccine immunized pigs.