猪流行性腹泻病毒N基因的表达及抗原性分析

Expression and antigenic analysis of porcine epidemic diarrhea virus N gene

为深入了解仔猪腹泻的病因,本研究在不同发病猪场采集了115份临床样品,通过RT-PCR方法对猪流行性腹泻病毒(PEDV)进行检测,结果显示总阳性率为81.7%。进一步从部分样品中对PEDV的N基因进行克隆和序列分析,结果显示采集的临床样品中各分离株之间的氨基酸同源性高达99%以上,与代表性病毒株CV777的氨基酸同源性为97.4%～98.1%,表明目前流行的PEDV其N基因仍然相对保守的。同时,构建了重组表达载体pGEX-N,经诱导表达显示该重组蛋白呈可溶性表达,重组蛋白大小约为81 ku,western blot分析结果表明该重组蛋白能够被PEDV阳性猪血清特异性识别。将纯化后的重组N蛋白作为包被抗原,对20份已知背景的临床猪血清抗体进行ELISA检测,结果显示有9份血清为抗体阳性,11份血清呈抗体阴性,与应用RT-PCR方法鉴定抗原的结果符合率为85%。本研究表达的重组N蛋白具有良好的抗原性,可以将其作为PEDV血清学诊断的候选抗原。

In recent two years, the outbreaks of porcine epidemic diarrhea epidemic caused heavy economic losses for pig industry in China. To investigate the epidemic of diarrhea, a total of 115 clinical samples were collected from different farms with occurrence of diarrhea and detected by RT-PCR, which showed an 81.7% positive rate for porcine epidemic diarrhea virus （PEDV）. In addition, nine of PEDV N genes were amplified and sequenced from positive samples, which exhibited that the homologies of deduced amino acid sequence were up to 99% among the isolates and 97.4% to 98.1% identity with PEDV CV777 reference strain. It indicated that N gene of the current PEDVs were still relatively conservative. Furthermore, the N gene was cloned into pGEX-6p-1 vector for expression in E.colL The expressed recombinant N protein was about 81 ku and reacted positively with PEDV serum. Moreover, twenty clinical pig sera, identified by RT-PCR, were detected by the ELISA coating with the recombinant PEDV N protein, and 17 samples were positive which were in line with the RT-PCR detection. In conclusion, these results indicated that the recombinant PEDV N protein might be a useful antigen for sera-diagnosis of PEDV infection in pigs.