基于猪丁型冠状病毒重组M蛋白间接ELISA抗体检测方法的建立与应用

Establishment and application of an indirect ELISA based on recombinant M protein for detection of the antibody against porcine deltacoronavirus

猪丁型冠状病毒(porcine deltacoronavirus, PDCoV)是近年新发现的一种猪肠道病原体,主要感染仔猪并引起仔猪腹泻甚至死亡,针对该病建立快速、准确的血清学检测方法尤为重要。本研究以PDCoV HNZK-04毒株为模板扩增M基因并将其克隆至原核表达载体pET-32a中,构建了重组质粒pET-32a-PDCoV-M。将阳性重组质粒转化至大肠杆菌BL21(DE3)中,并用IPTG诱导M蛋白表达,SDS-PAGE显示目的蛋白的分子量约为41 kDa;Western blot结果显示该蛋白可以与PDCoV阳性血清特异性结合。以纯化后的M蛋白作为靶抗原,建立了一种间接ELISA检测方法。用该方法对其他猪常见病毒,包括猪流行性腹泻病毒(PEDV)、猪传染性胃肠炎病毒(TGEV)、猪伪狂犬病病毒(PRV)、猪繁殖与呼吸综合征病毒(PRRSV)和猪圆环病毒2型(PCV2)的阳性血清进行检测,结果均为阴性;批内重复试验和批间重复试验变异系数均小于10%,表明建立的PDCoV间接ELISA检测方法具有良好的特异性和重复性,可以用于PDCoV感染的临床检测和诊断。

Porcine deltacoronavirus(PDCoV) is a newly discovered porcine enteric pathogen in recent years, and it mainly causes diarrhea and even death in piglets. Therefore, it is important to establish a rapid and accurate serological detection method for PDCoV. In this study, the M gene was amplified using a PDCoV HNZK-04 strain as a template and cloned into the prokaryotic expression vector pET-32 a to construct a recombinant plasmid pET-32 a-PDCoV-M. The positive recombinant plasmid was transformed into E. coli BL21(DE3) and the expression of the M protein was induced by IPTG. The results were as follows: SDS-PAGE showed that the molecular weight of the expressed protein was about 41 kDa. Western blot analysis showed that the protein reacted specifically with PDCoV positive serum. Then, the protein was purified and coated as the target antigen to establish an indirect ELISA assay. The specificity test showed that the antigen had no cross reaction with positive serum of other porcine viruses, including PEDV, TGEV, PRV, PRRSV and PCV2. The intra-and inter-assay variation coefficients were less than 10%. The above results indicated that the PDCoV indirect ELISA established here had good specificity and reproducibility, which was applicable to the detection and diagnosis of PDCoV.