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  

Establishment of an indirect enzyme-linked immuno-sorbent assay for detecting PRRSV serum antibody based on tandem expression with antigen epitopes of GP5 and N proteins

作　　者：陈如敬[1] 周伦江[1] 吴学敏[1] 车勇良[1] 王晨燕[1] 王隆柏[1] 黄晓凤[1] 严山[1] 刘玉涛[1] 魏宏[1] CHEN Rujing ZHOU Lunjiang WU Xuemin CHE Yongliang WANG Chenyan WANG Longbai HUANG Xiaofeng YAN Shan LIU Yutao WEI Hong(Fujian Animal Diseases Control Technology Development Center/Institute of Animal Husbandry and Veterinary Medicine, Fujian Academy of Agricultural Sciences, Fuzhou, Fuj Jan 350013, China)

机构地区：[1]福建省农业科学院畜牧兽医研究所/福建省畜禽疫病防治工程技术研究中心,福建福州350013

出　　处：《西北农林科技大学学报（自然科学版）》2017年第10期15-20,29,共7页Journal of Northwest A&F University(Natural Science Edition)

基　　金：福建省属公益类项目(2014R1023-16;2014R1023-13);福建省畜禽疫病防控技术重大研发平台项目(2014N2003)

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,OD450<0.345 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。【Objective】 This study established an indirect enzyme-linked immuno-sorbent assay（ELISA）to detect the serum antibody levels of porcine reproductive and respiratory syndrome virus（PRRSV）based on tandem expression with the antigen epitopes of GP5 and N proteins to provide basis fordeveloping accurate and inexpensive PRRSV serological test kits.【Method】After the prokaryotic tandem expression of N and GP5 proteins antigen epitopes of PRRSV,the expressed proteins were purified for use as coating antigen to determine the best indirect ELISA conditions.Then its specificity and repeatability were detected.Forty-five clinical serum samples were tested by the established ELISA and the coincidence rate was calculated by comparing with commercial IDEXX kit.【Result】SDS-PAGE and Western Blot analysis showed the weight of the recombinant soluble protein was 24.7ku with good biological activity.The target proteins were purified and used as coating antigen to establish an indirect ELISA with following optimal conditions：coating concentration of purified proteins was 5μg/mL,the serum was diluted by 1∶80,the enzyme labeled antibody was diluted by 1∶5 000,using 50g/L skim milk powder in PBST as sealing fluid for 60 min at 37 ℃,antibody（the serum and enzyme labeled antibody）were incubated for 90 min at37 ℃,and the reaction time of substrate was 8min protected from light.The cut-off value of the assay was 0.345 2,with OD450≥0.345 2for positive and OD450〈0.345 2for negative.No cross-reactivity was observed with common porcine viruses＇ hyper immune serum,such as pseudorabies virus,porcine circovirus type 2,classic swine fever virus and porcine Japanese encephalitis virus.Reproducibility test showed that the intra-and inter-assay were 1.02%-3.94%and 1.38%-4.83%,respectively.Forty-five clinical serum samples were tested by the established ELISA and the commercial IDEXX kit and their positive rates were84.44%（38/45）and 80%（36/45）,respectively.The IDEXX kit positive samples were tested positi

关 键 词：猪繁殖与呼吸综合征病毒 N蛋白 GP5蛋白 抗原表位 串联表达 间接ELISA 

分 类 号：S858.28[农业科学—临床兽医学]