摘要
通过DNAStar Protean软件对猪德尔塔冠状病毒(porcine Deltacoronavirus,PDCoV)N蛋白氨基酸序列进行分析,选取抗原性较强的区段(265~342 aa),根据大肠杆菌密码子使用的偏嗜性进行优化设计并人工合成了对应的基因片段N-Opti。利用融合表达载体pET-32a进行原核表达,获得的融合蛋白携带His标签,命名为PDCoV-N-His,且呈可溶性表达。Western blot鉴定结果显示,该重组N蛋白约为31 kDa,与预期相符。利用Ni-NTA亲和层析介质纯化重组N蛋白作为包被抗原进行ELISA检测,并对反应条件进行摸索、优化,建立了检测PDCoV特异性抗体的间接ELISA方法。经测定,该方法具有良好的特异性且重复性良好,与猪流行性腹泻病毒(PEDV)、猪传染性胃肠炎病毒(TGEV)和猪轮状病毒(PRoV)特异性阳性血清无交叉反应,批内和批间重复性试验的变异系数均小于10%。应用该方法对20份已知阳性血清和20份阴性血清分别进行检测,结果显示阳性符合率为100%(20/20),阴性符合率为100%(20/20)。结果表明,该方法的特异性良好,可以进一步用于PDCoV感染的快速诊断和流行病学调查。
The amino acid sequence of porcine Deltacoronavirus(PDCoV)N protein was analyzed by DNAStar Protean software,and the region with strong antigenicity(265-342 aa)was selected.According to the frequency of E.coli codons,the optimized design was carried out and the corresponding gene fragment N-opti was artificially synthesized and cloned into pET32 a for the prokaryotic expression of N protein.The obtained fusion protein was labeled with His-tag and named as PDCoV-N-His,it was a soluble protein.The Western blot results showed that the size of the recombinant N protein was about 31 kDa.The recombinant N protein was purified by Ni-NTA affinity chromatography and used as the coating antigen.By optimizing the reaction conditions,an indirect ELISA method for detection of PDCoV specific antibody was established.The results showed that this indirect ELISA had good specificity and repeatability,since it has no cross-reaction with the positive serum of PEDV,TGEV and PRoV,the variation coefficient of the inter-batch and intra-batch repeatability test was less than 10%.The indirect ELISA was used to detect 20 PDCoV positive serum and 20 negative serum,and the results showed that the positive and negative coincidence rate was both 100%.The results showed that the indirect ELISA for PDCoV antibodies has good specificity and can be used for rapid diagnosis and epidemiological investigation of PDCoV infection.
作者
钱炳旭
白雪雁
张成成
郭梦娇
李梦娇
廖凯
薛峰
吴艳涛
张小荣
QIAN Bingxu;BAI Xueyan;ZHANG Chengcheng;GUO Mengjiao;LI Mengjiao;LIAO Kai;XUE Feng;WU Yantao;ZHANG Xiaorong(Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses,College of Veterinary Medicine,Yangzhou University,Yangzhou,Jiangsu 225000,China;International Cooperative Laboratory for Animal Health and Food Safety,Nanjing Agricultural University,Nanjing 210095 China)
出处
《中国兽医学报》
CAS
CSCD
北大核心
2021年第7期1234-1241,共8页
Chinese Journal of Veterinary Science
基金
扬州大学高端人才支持计划资助项目(2016年)
江苏高校优势学科建设工程资助项目(2018年)。
