Postweaning multisystemic wasting syndrome (PMWS) is an important swine disease that is closely associated with porcine circovirus type 2 (PCV2). The capsid protein (Cap protein) is a major structural protein that has...Postweaning multisystemic wasting syndrome (PMWS) is an important swine disease that is closely associated with porcine circovirus type 2 (PCV2). The capsid protein (Cap protein) is a major structural protein that has at least three immunoreactive regions, and it can be a suitable candidate antigen for detecting the specific antibodies of a PCV2 infection. In the present study, an indirect enzyme-linked immunosorbent assay (TcELISA) based on a truncated soluble Cap protein produced in Escherichia coli (E.coli) was established and validated for the diagnostic PCV2 antibodies in swine. The TcELISA was validated by comparison with an indirect immunofluorescence assay (IIFA). The diagnostic sensitivity (DSN), specificity (DSP), and accuracy of the TcELISA were 88.6%, 90.7% and 89.4%, respectively. The agreement rate was 89.38% between results obtained with TcELISA and IIFA on 113 field sera. A cross-reactivity assay showed that the method was PCV2-specific by comparison with other sera of viral disease. Therefore ,the TcELISA will be helpful for the development of a reliable serology diagnostic test for large scale detection of PCV2 antibodies and for the evaluation of vaccine against PCV2 in swine.展开更多
基金supported by a project from National Key Technology R&D Program in the 11th Fiveyear Plan of China (2006BAD06A12)
文摘Postweaning multisystemic wasting syndrome (PMWS) is an important swine disease that is closely associated with porcine circovirus type 2 (PCV2). The capsid protein (Cap protein) is a major structural protein that has at least three immunoreactive regions, and it can be a suitable candidate antigen for detecting the specific antibodies of a PCV2 infection. In the present study, an indirect enzyme-linked immunosorbent assay (TcELISA) based on a truncated soluble Cap protein produced in Escherichia coli (E.coli) was established and validated for the diagnostic PCV2 antibodies in swine. The TcELISA was validated by comparison with an indirect immunofluorescence assay (IIFA). The diagnostic sensitivity (DSN), specificity (DSP), and accuracy of the TcELISA were 88.6%, 90.7% and 89.4%, respectively. The agreement rate was 89.38% between results obtained with TcELISA and IIFA on 113 field sera. A cross-reactivity assay showed that the method was PCV2-specific by comparison with other sera of viral disease. Therefore ,the TcELISA will be helpful for the development of a reliable serology diagnostic test for large scale detection of PCV2 antibodies and for the evaluation of vaccine against PCV2 in swine.
文摘【目的】由新型鹅星状病毒(novel goose astrovirus,nGAstV)引起的雏鹅痛风病对我国养鹅业造成了重大经济损失。通过构建nGAstV纳米抗体(nanobody,Nb)噬菌体展示文库,获得识别nGAstV ORF2蛋白的特异性Nb,可为建立nGAstV抗体检测方法及研究nGAstV ORF2蛋白结构和功能奠定基础。【方法】使用蔗糖密度梯度离心方法纯化在LMH细胞中增殖的nGAstV,RT-PCR鉴定nGAstV并通过细胞病变测定nGAstV滴度。用0.06%甲醛溶液灭活纯化的nGAstV作为免疫原,免疫两周岁羊驼。首次免疫时用灭活纯化的nGAstV与等量弗氏完全佐剂乳化后免疫,第2—5次免疫用灭活纯化的nGAstV与等量弗氏不完全佐剂乳化。每间隔两周免疫一次,每次免疫剂量均为50μg。第5次免疫14 d后,通过间接ELISA方法测定羊驼血清中针对nGAstV的IgG抗体效价。待IgG抗体效价达到构建噬菌体抗体展示文库标准时,分离羊驼外周血淋巴细胞(peripheral blood lymphocyte,PBL),然后提取PBL总RNA将其反转录为cDNA,利用巢氏PCR方法扩增重链抗体重链可变区(variable domain of the heavy chain of heavy chain antibodies,VHH)基因,将其构建至pComb噬菌体载体并结合噬菌体展示技术构建nGAstV Nb噬菌体展示文库,计算该文库库容量并分析其多样性。nGAstV作为靶抗原,通过三轮富集淘选初步获得与nGAstV反应的重组噬菌体Nb阳性克隆,将其克隆至pcDNA3.1-Fc真核表达载体并进行测序分析,将测序成功且序列不同的质粒转染至HEK-293F细胞中表达,并通过聚丙烯酰胺凝胶电泳(SDS-PAGE)鉴定表达情况。以nGAstV为靶标抗原,利用间接ELISA方法和Western blot试验对表达成功的Nb进行特异性、反应活性验证,并以nGAstV ORF2蛋白为靶标抗原,利用间接ELISA方法对Nb进行亲和力验证,获得生物学活性较好的Nb。【结果】RT-PCR结果显示,nGAstV增殖成功;Reed-Muench法分析nGAstV滴度达4.38 Log10TCID50/mL。羊驼经5次免疫nGAstV后,通过间接ELISA方法测得其血清中针对nGAstV的抗体效价达到1:64000;利用巢氏PCR方法扩增出VHH并成功构建库容量为3.8×108CFU/mL的nGAstV Nb噬菌体展示文库;遗传进化树分析显示,该噬菌体Nb库多样性良好。三轮富集淘选后,初步获得39株与nGAstV反应的重组噬菌体纳米抗体阳性克隆,其中有25株序列不同;SDS-PAGE鉴定结果显示,共有10株Nb在HEK-293F细胞中表达成功。通过ELISA、Western blot验证进一步获得8株与nGAstV ORF2蛋白特异性反应的Nb,且1株Nb生物学活性最好。【结论】首次筛选到与nGAstV ORF2蛋白特异性反应的Nb,为nGAstV的基础研究和检测方法提供材料。
