H1亚型猪流感病毒抗体间接ELISA检测方法的建立及其在浙江省的应用  

Development of indirect ELISA for detection of antibody against swine influenza virus H1 subtype and its application in Zhejiang Province

作　　者：颜焰[1] 曾嘉梅[1] 周继勇[1] 廖敏[1] 

机构地区：[1]浙江大学农业部动物病毒学重点实验室,浙江杭州310058

出　　处：《中国兽医科学》2015年第11期1101-1107,共7页Chinese Veterinary Science

基　　金：国家科技支撑计划项目(2010BAD04B00);浙江大学自主科研计划项目(2015FZA6017)

摘　　要：利用RT-PCR扩增了猪流感病毒分离株A/Swine/Zhejiang/103/2002(H1N1)HA1基因,并将其克隆至pFastBacHTA杆状病毒转移载体。将筛选的阳性重组转移载体pFastBacHTA-HA1转化至含有杆状病毒穿梭载体的DH10Bac感受态细胞,构建重组转座子rBacmid-HA1。之后转染Sf9昆虫细胞制备rBV-HA1重组杆状病毒。SDS-PAGE分析显示,重组表达的HA1蛋白的分子质量约为45ku。Westernblot结果显示,该HA1蛋白能与H1亚型猪流感阳性血清特异性结合,具有良好的抗原活性。利用镍柱纯化后的HA1蛋白作为包被抗原,建立了H1亚型猪流感病毒抗体间接ELISA检测方法,通过对各种条件进行优化,确定了最佳反应体系。该方法与多种猪病毒阳性血清无交叉反应性,与HI方法的符合率为92.4%,与IDEXX进口试剂盒的符合率为94.6%。用该方法对2014年从浙江省规模化猪场采集的猪血清进行检测,阳性率为17.4%(589/3 379)。该方法的建立为浙江省猪流感的监测及防控提供了重要检测工具。The HA1 gene of the swine influenza virus（SIV）A/Swine/Zhejiang/103/2002（H1N1）was amplified by reverse transcription PCR,and cloned into the baculovirus transfer vector pFastBacHTA,named as pFastBacHTA-HA1.The recombinant plasmid pFastBacHTA-HA1 was then transferred into DH10 Bac cells contained shuttle plasmid.The rBacmid-HA1 plasmid was then transfected into the insect Sf9 cells.SDS-PAGE assay showed that the relative molecular weight of recombinant HA1 protein is 45 ku.Western-blot assay demonstrated that the recombinant HA1 protein could be recognized by the antiSIV/H1 positive serum.Based on the purified HA1 protein,an indirect enzyme-linked immunosorbent assay（ELISA）with optimized condition for detection of antibody against swine influenza virus H1 subtype was developed.The result showed no cross-reaction with other positive sera against swine viruses.The coincidence rates between indirect ELISA with HI test and IDEXX kit were 92.4%and 94.6%,respectively.A total of 3 379 serum samples from Zhejiang large-scale commercial swine herds in 2014 were detected through this method,589 of which were positive and the positive rate was 17.4%.In conclusion,the indirect ELISA provided a useful tool for investigation and prevention of SIV in Zhejiang Province.

关 键 词：猪流感病毒 血凝素抗原 杆状病毒 间接ELISA 抗体检测 

分 类 号：S852.659.5[农业科学—基础兽医学]