猪繁殖与呼吸综合征病毒截短GP5蛋白间接ELISA方法的建立  

Establishment of an indirect ELISA method for detection of antibody against PRRSV with truncated GP5 protein

作　　者：陈攀[1] 郑芳园[1] 李玉峰[1] 

机构地区：[1]南京农业大学农业部细菌学重点开放实验室,江苏南京210095

出　　处：《畜牧与兽医》2014年第4期36-40,共5页Animal Husbandry & Veterinary Medicine

基　　金：公益性行业(农业)科研专项项目(200903036-10)

摘　　要：猪繁殖与呼吸综合征病毒(PRRSV)感染主要引起母猪繁殖障碍和新生仔猪呼吸道症状,目前尚无有效的血清学检测方法评价猪群疫苗免疫或感染后抗体水平与免疫保护力之间的关系。为建立PRRSV GP5蛋白的ELISA方法,本研究选择GP5蛋白亲水区进行原核表达,以表达的重组蛋白tGP5为包被抗原建立了检测针对GP5蛋白抗体的ELISA方法,优化后反应条件为:抗原包被浓度2μg/mL,37℃包被2 h,5%脱脂乳37℃封闭2 h,待检血清稀释度1∶100,37℃作用1 h,二抗1∶20 000稀释,37℃作用45 min,37℃显色3 min,抗体临界值OD450nm≥0.22判为阳性,OD450nm<0.183判为阴性,介于两者之间为可疑。特异性和重复性试验证明,与猪瘟病毒、猪伪狂犬病毒、猪圆环病毒2型、猪口蹄疫病毒血清抗体无交叉反应,批内、批间重复性较好。Porcine reproductive and respiratory syndrome virus （PRRSV） can cause reproductive failures in sows and respiratory problems in piglets. There are no effective serological methods to assess the relationship between antibody levels after vaccination and immune efficacy. To evaluate the relationship, an indirect ELISA was established for detection of antibody against this virus using the recombinant truncated GP5 （tGP5） protein as coating antigen. The optimized reaction conditions were included： coating with 2 μg/mL antigen at 37 ℃ for 2 hours, blocking with 5% skim milk at 37 ℃ for 2 hours, incubating with sera diluted at 1 ： 100 at 37 ℃ for one hour, then incubating with the secondary antibody diluted at 1 ： 20 000 at 37 ℃ for 45 minutes, and judging with the cutoff value of OD450mm 90. 22. The analysis for specificity and reproducibility showed that it had no reactions with the positive sera to classical swine fever virus, pseudorabies virus, porcine circovirus type 2 virus and foot-and-mouth disease virus, and the inter-and intra-bateh reproducibility was good.

关 键 词：PRRSV 重组蛋白tGP5 原核表达 间接ELISA 

分 类 号：S855.3[农业科学—临床兽医学]