一种基于猪流行性腹泻病毒S1蛋白的单域抗体的阻断ELISA方法及其评价

Development of a blocking ELISA based on a single-domain antibody target the S1 protein of porcine epidemic diarrhea virus

文中旨在利用猪流行性腹泻病毒(Porcine epidemic diarrhea virus,PEDV)S1蛋白生物素化纳米抗体建立一种阻断酶联免疫吸附试验(Blocking enzyme-linked immunosorbent assay,bELISA)方法,用于检测猪体内PEDV抗体水平及疫苗免疫效果的评估。对PEDVS1蛋白的特异性单域抗体(Single-domain antibodies,sdAb)sdAb3基因进行扩增,并在3′端融合Avitag序列,构建至原核表达载体pET21b,进行sdAb3-Avitag蛋白诱导表达纯化。对纯化的sdAb3-Avitag融合蛋白进行生物素标记并鉴定其活性。以PEDV重组S1蛋白为抗原,通过对各反应条件进行摸索与优化,建立一种可靠灵敏的bELISA方法应用于血清样品检测,并与商品化试剂盒检测进行比价。成功构建重组载体pET21b-sdAb3-Avitag并诱导表达出sdAb3-Avitag蛋白。体外生物素标记的sdAb3(sdAb3-Biotin)具有良好的活性。所构建的bELISA方法中,最适参数为:S1蛋白的包被浓度200 ng/孔;血清稀释比例1︰2,血清孵育时间2 h;sdAb3-Biotin稀释比1︰8000,孵育时间30 min;酶标抗体稀释比例1︰5000,反应时间30 min。所建立的方法与猪传染性胃肠炎病毒、猪繁殖与呼吸综合征病毒等主要猪源病毒的阳性血清均无交叉反应,具有良好的特异性和重复性。利用建立的bELISA方法对临床54份猪血清样品进行检测,结果显示,该方法与商品化试剂盒检测结果具有92.56%的总体符合率。文中建立了一种省时可靠的bELISA方法,可用于PEDV的临床监测和疫苗免疫效果评估。

The aim of this study was to develop a blocking enzyme-linked immunosorbent assay(bELISA) based on a biotinylated nanobody target the S1 protein of porcine epidemic diarrhea virus(PEDV) for detecting the anti-PEDV antibodies and evaluating the immune effect of the vaccine. The gene encoding the single-domain antibody sdAb3 target the PEDV S1 protein was amplified and the Avitag sequence was fused at its 3′-end. The PCR product was cloned into the expression vector pET-21 b for expression and purification of the sdAb3-Avitag protein. The purified sdAb3-Avitag fusion protein was biotinylated and its activity was determined. Using the recombinant S1 protein as a coating antigen, a bELISA was established and optimized. Serum samples were tested in parallel by the bELISA and a commercial kit. The recombinant vector pET21 b-sdAb3-Avitag was constructed to express the tagged sdAb3. After induction for expression, the biotin-labeled sdAb3(sdAb3-Biotin) with high purity and good activity was obtained. For the optimized bELISA, the coating concentration of the S1 protein was 200 ng/well, the serum dilution was 1:2 and incubated for 2 h, the dilution ratio of the biotinylated sdAb3 was 1:8 000 and incubated for 30 min, the dilution of the enzyme-labeled antibody was 1:5 000 and incubated for 30 min. The bELISA had no cross reaction with the sera of major porcine viruses including transmissible gastroenteritis virus, porcine reproductive and respiratory syndrome virus and showed good specificity and reproducibility. For a total of 54 porcine serum samples tested, the overall compliance rate of the bELISA with a commercial kit was 92.56%. This study developed a rapid and reliable bELISA method, which can be used for serosurveillance and vaccine evaluation for PEDV.