基于猪流行性腹泻病毒变异毒株重组全长S2蛋白IgA抗体ELISA检测方法的建立及应用  

Establishment and Application of ELISA for Detection of IgA Antibody Based on the Recombinant Full-length S2 Protein of Porcine Epidemic Diarrhea Virus Variant Strain

作　　者：牟一娇 于瑞明 张莉萍 王永录[2] MU Yijiao;YU Ruiming;ZHANG Liping;WANG Yonglu(China Agricultural Vet Biologyand Technology Co.,Ltd.,Lanzhou 730046,China;Lanzhou Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Lanzhou 730046,China)

机构地区：[1]中农威特生物科技股份有限公司,兰州730046 [2]中国农业科学院兰州兽医研究所,兰州730046

出　　处：《中国畜牧兽医》2023年第3期1185-1194,共10页China Animal Husbandry & Veterinary Medicine

基　　金：国家生猪产业体系项目(CARS-35);兰州兽医研究所所级基本科研业务费(1610312021011)。

摘　　要：【目的】建立一种猪流行性腹泻病毒(Porcine epidemic diarrhea virus,PEDV)的IgA抗体ELISA检测方法。【方法】以PEDV流行株CH/HNPJ/2017(MF152604.1)为生物材料,通过RT-PCR扩增出S 2基因(2368—4170 bp),并将其插入到原核表达载体pET-24a中,转化大肠杆菌BL21(DE3)感受态细胞,利用IPTG诱导表达,经镍柱纯化。Western blotting验证重组蛋白S2与PEDV阳性血清的反应原性和特异性。将其作为包被抗原,建立一种基于PEDV变异毒株重组全长S2蛋白的IgA抗体ELISA方法,用棋盘法确定该方法的最优检测条件,对其敏感性、特异性和重复性进行测定,并初步应用于130份猪血清和40份猪口腔黏液的检测。【结果】通过RT-PCR方法扩增出S 2基因,成功构建重组表达载体pET-24a-S2,转化大肠杆菌经诱导表达纯化后获得重组S2蛋白。SDS-PAGE结果显示,纯化的S2蛋白条带特异且无杂带;Western blotting试验表明该蛋白与PEDV阳性猪血清具有良好的反应性。ELISA方法反应条件优化结果显示,重组S2蛋白的最佳包被条件为每孔0.5μg、4℃过夜包被;血清最佳反应条件为1∶160稀释,37℃孵育60 min;酶标二抗最佳反应条件为1∶5000稀释,37℃反应30 min;TMB最佳显色时间为室温下10 min。待检样品S/P值≥0.04时为阳性,S/P值≤0.02时为阴性,S/P值介于0.02~0.04之间为可疑。利用本试验建立的ELISA方法检测猪瘟病毒、猪圆环病毒、猪繁殖与呼吸综合征病毒、伪狂犬病病毒、猪细小病毒、猪德尔塔冠状病毒标准阳性血清,S/P值均<0.02,显示为阴性,说明该方法具有良好的特异性;将PEDV阳性血清进行640倍稀释时利用该方法检测仍为阳性,说明该方法具有良好的敏感性;利用该方法进行批间批内试验,变异系数均小于8%(在10%以内),表明该方法具有良好的重复性。利用该方法检测保存的130份猪血清,结果与IDEXX IgA抗体检测试剂盒的符合率为90.77%;该方法对40份猪口腔黏液的�【Objective】The purpose of this study was to establish an ELISA method for detection of IgA antibody against Porcine epidemic diarrhea virus(PEDV).【Method】In this study,PEDV epidemic strain CH/HNPJ/2017(MF152604.1)was used as the biological material,and the S 2 gene(2368—4170 bp)was amplified by RT-PCR and inserted into prokaryotic expression vector pET-24a to transform the competent cells of Escherichia coli BL21(DE3).It was induced by IPTG and purified by nickel column.Western blotting was used to verify the reactivity and specificity of recombinant protein S2 with PEDV positive serum.Using it as the coating antigen,an IgA antibody ELISA method based on recombinant full-length S2 protein of PEDV variant strain was established.The chessboard method was used to determine the optimal detection conditions,and its sensitivity,specificity and repeatability were determined.It was preliminarily applied to the detection of 130 porcine serum and 40 porcine oral mucus.【Result】The S 2 gene was amplified by RT-PCR,and the recombinant expression vector pET-24a-S2 was successfully constructed.The recombinant S2 protein was obtained after being transformed into Escherichia coli and expressed and purified by induction.SDS-PAGE results showed that the purified S2 protein had specific bands and no heterobands.Western blotting test showed that the protein had good reactivity with PEDV positive pig serum.The optimal coating conditions for recombinant S2 protein were 0.5μg per well at 4℃overnight,the optimal serum reaction conditions were 1∶160 dilution at 37℃for 60 min,the best reaction conditions for HRP-conjugated antibody were 1∶5000 dilution at 37℃for 30 min,and the optimal color rendering time of TMB was 10 min under ambient temperatures.The sample was positive when the S/P value≥0.04,it was negative when the S/P value≤0.02,and it was suspicious when the S/P value was between 0.02 and 0.04.The ELISA method established in this study was used to detect the standard positive serum of swine fever virus,

关 键 词：猪流行性腹泻 S2蛋白 IGA抗体 ELISA 

分 类 号：S852.53[农业科学—基础兽医学]