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当前位置: 首页 > 文章 > 以PRRSV Nsp2-399重组蛋白为包被抗原建立PRRSV抗体ELISA检测方法 黑龙江畜牧兽医 2019 (16) 76-80
Position: Home > Articles > 以PRRSV Nsp2-399重组蛋白为包被抗原建立PRRSV抗体ELISA检测方法 Heilongjiang Animal Science and Veterinary Medicine 2019 (16) 76-80

以PRRSV Nsp2-399重组蛋白为包被抗原建立PRRSV抗体ELISA检测方法

作  者：
修晓娜;张松林;沈志强;刘磊;马永彪
单  位：
中国农业大学烟台研究院
关键词：
PRRSV;非结构蛋白2;抗体检测;Nsp2-399;ELISA抗体
摘  要：
为了获得猪繁殖与呼吸综合征病毒(PRRSV)Nsp2-399重组蛋白,以及利用Nsp2-399蛋白建立PRRSV早期感染的ELISA检测方法,试验克隆、原核表达了PRRSV DY株(GenBank登录号为JN864948)Nsp2-399重组蛋白,同时利用该蛋白建立并优化了ELISA检测方法.结果表明:成功克隆表达了PRRSV Nsp2-399重组蛋白,并建立、优化了Nsp2-399 ELISA检测方法.优化后的Nsp2-399 ELISA的最佳抗原包被浓度为1μg/mL,血清最佳稀释度为1∶40,最佳封闭液为10%胎牛血清,酶标二抗的最佳工作稀释度为1∶4 000,最佳显色条件为37℃、15 min.猪伪狂犬病毒、猪瘟病毒、猪圆环病毒、猪细小病毒阳性血清样品用建立的Nsp2-399 ELISA方法进行检测,S/P值均小于0.21,ELISA方法特异性良好.用本试验建立的ELISA方法和IDEXX ELISA检测试剂盒分别检测血清样品125份,总符合率为94.40%.说明成功建立了Nsp2-399 ELISA抗体检测方法,可用于PRRSV的抗体检测.
作  者：
XIU Xiaona;ZHANG Songlin;SHEN Zhiqiang;LIU Lei;MA Yongbiao;Yantai Institute, China Agricultural University;Shandong Binzhou Animal Science & Veterinary Medicine Academy;Shandong Lvdu Bio-technology Co.Ltd.;
单  位：
XIU Xiaona%ZHANG Songlin%SHEN Zhiqiang%LIU Lei%MA Yongbiao%Yantai Institute, China Agricultural University%Shandong Binzhou Animal Science & Veterinary Medicine Academy%Shandong Lvdu Bio-technology Co.Ltd.
关键词：
PRRSV;;non-structural proteins 2;;antibody detection;;Nsp2-399;;ELISA antibody
摘  要：
In order to obtain the recombinant protein of PRRSV Nsp2-399 and establish an ELISA method for detection of early infection of PRRSV, Nsp2 gene was cloned and expressed from PRRSV DY strain(GenBank: JN864948), and a Nsp2-ELISA detection method was established and optimized by using Nsp2 protein in this study. The results showed that the recombinant protein of PRRSV Nsp2-399 was cloned and expressed successfully, and the detection method of Nsp2-399 ELISA was established and optimized. For Nsp2-399 ELISA, optimal antigen package was 1 μg/mL, the best dilution of the serum was 1∶40, the best closed liquid was 10% bovine serum, the best working dilution of enzyme labeled secondary antibody was 1∶4 000, and the best color condition was 37 ℃, 15 min.The samples of porcine pseudorabies virus, classical swine fever virus, porcine circovirus and porcine parvovirus positive serum were detected by the Nsp2-399 ELISA method.The S/P values were less than 0.21,and the ELISA method was very specific. For compliance experiment, 125 serum samples were detected by IDEXX ELISA Kit, and the total compliance rate was 94.40%. The establishment of Nsp2-399 ELISA antibody detection method provides a new idea for PRRSV antibody monitoring.

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