PRRSV N与GP5蛋白抗原表位的串联表达及其间接ELISA检测方法的建立
发布时间：2018-10-15 15:07
【摘要】：【目的】建立基于N与GP5蛋白抗原表位串联的猪繁殖与呼吸综合征病毒(Porcine reproductive and respiratory syndrome virus,PRRSV)抗体ELISA检测方法,为开发准确、廉价的PRRSV抗体检测试剂盒奠定基础。【方法】将PRRSV的N蛋白和GP5蛋白抗原表位串联优化后进行原核表达,以表达的目的蛋白为抗原,通过优化反应条件建立检测PRRSV抗体的ELISA方法,对其特异性、重复性进行检测。用建立的间接ELISA方法和商品化IDEXX试剂盒同时对临床送检的45份猪血清样品进行检测,计算其符合率。【结果】SDS-PAGE和Western Blot分析表明,表达的目的蛋白分子质量约为24.7ku,具有良好的生物学活性,且为可溶性表达。目的蛋白经纯化后作为抗原包被ELISA平板,建立检测PRRSV抗体的间接ELISA方法,优化后的ELISA条件为:抗原(纯化后的目的蛋白)包被量为5μg/mL、一抗(猪血清)1∶80稀释、酶标二抗1∶5 000稀释,以含50g/L脱脂奶粉的PBST作为封闭液,37℃孵育60min,抗体(一抗和酶标二抗)于37℃孵育90min,TMB避光显色8min;间接ELISA的判定标准为:OD450≥0.345 2为阳性,OD4500.345 2为阴性。所建立的间接ELISA方法特异性强,对猪常见病原,如猪伪狂犬病毒、猪圆环病毒2型、猪瘟病毒和猪流行性乙型脑炎病毒高免血清检测均为阴性;重复性好,组内变异系数和组间变异系数分别为1.02%~3.94%和1.38%~4.83%。用所建立的间接ELISA方法对临床送检45份猪血清样品的检测阳性率为84.44%(38/45),商品化IDEXX试剂盒检测的阳性率为80%(36/45),2种方法的符合率为94.73%(36/38)。【结论】成功建立了基于N与GP5蛋白抗原表位串联的PRRSV抗体ELISA检测方法。
[Abstract]:[objective] to establish a ELISA method for detection of porcine reproductive and respiratory syndrome virus (Porcine reproductive and respiratory syndrome virus,PRRSV) antibody based on N and GP5 epitopes. The cheap PRRSV antibody detection kit laid the foundation. [methods] the N protein of PRRSV and the antigen epitope of GP5 protein were optimized for prokaryotic expression, and the expressed target protein was used as antigen. A ELISA method for detection of PRRSV antibody was established by optimizing the reaction conditions, and its specificity and repeatability were detected. The indirect ELISA method and commercial IDEXX kit were used to detect 45 pig serum samples for clinical examination, and the coincidence rate was calculated. [results] SDS-PAGE and Western Blot analysis showed that, The molecular weight of the expressed target protein is about 24.7ku. it has good biological activity and soluble expression. Objective protein was purified as antigen to coat ELISA plate, and an indirect ELISA method for detection of PRRSV antibody was established. The optimized ELISA conditions were as follows: the amount of antigen (purified target protein) was 5 渭 g / mL, and the first antibody (pig serum) was diluted at 1:80. The enzyme labeled second antibody was diluted at 1:5, PBST containing 50g/L skim milk powder was incubated at 37 鈩，
本文编号：2272925
[Abstract]:[objective] to establish a ELISA method for detection of porcine reproductive and respiratory syndrome virus (Porcine reproductive and respiratory syndrome virus,PRRSV) antibody based on N and GP5 epitopes. The cheap PRRSV antibody detection kit laid the foundation. [methods] the N protein of PRRSV and the antigen epitope of GP5 protein were optimized for prokaryotic expression, and the expressed target protein was used as antigen. A ELISA method for detection of PRRSV antibody was established by optimizing the reaction conditions, and its specificity and repeatability were detected. The indirect ELISA method and commercial IDEXX kit were used to detect 45 pig serum samples for clinical examination, and the coincidence rate was calculated. [results] SDS-PAGE and Western Blot analysis showed that, The molecular weight of the expressed target protein is about 24.7ku. it has good biological activity and soluble expression. Objective protein was purified as antigen to coat ELISA plate, and an indirect ELISA method for detection of PRRSV antibody was established. The optimized ELISA conditions were as follows: the amount of antigen (purified target protein) was 5 渭 g / mL, and the first antibody (pig serum) was diluted at 1:80. The enzyme labeled second antibody was diluted at 1:5, PBST containing 50g/L skim milk powder was incubated at 37 鈩，
本文编号：2272925
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