猪流感病毒HA基因的原核表达及其ELISA检测方法的建立  

Expression of Hemagglutinin Gene of Swine influenza virus and Establishment of ELISA for Serum Detection

作　　者：李春[1] 宋云峰[1] 金梅林[1] 陈焕春[1] 

机构地区：[1]华中农业大学动物医学院农业微生物国家重点实验室,湖北武汉430070

出　　处：《动物医学进展》2007年第7期35-39,共5页Progress In Veterinary Medicine

摘　　要：根据H1N1亚型猪流感病毒血凝素基因序列设计并合成特异性引物,从本室保存的H1N1亚型猪流感病毒中扩增信号肽和跨膜区缺失的血凝素基因,并将其克隆到原核表达载体pET-28a中,在大肠埃希菌中诱导表达。对表达蛋白进行SDS-PAGE和Western blot的结果表明,HA基因在大肠埃希菌中获得表达,产物具有免疫学活性,表达蛋白分子质量约为55 ku。表达产物经过纯化作为包被抗原建立了检测H1亚型猪流感抗体的间接ELISA方法。该方法具有较高的特异性和敏感性,重复性良好。应用该方法对2006年2 584份临床猪血清进行检测,结果显示多个地区检测猪流感抗体出现阳性,平均为20.5%。在6月、7月和12月分别出现抗体水平高峰,分别高达25.4%、25.0%和35.5%。The HA gene of swine influenza virus（SIV） H1 subtype was cloned, expressed and used for an enzyme-linked immunity sorbent assay（ELISA） to diagnose SIV H1 subtyes. A part of HA gene without signal sequence and transmembrance sequence of H1N1 SIV was amplified from a strain of the field isolated H1N1 SIV, and then the modified HA gene was subcloned into pET-28a. The expressed HA protein was identified by SDS-PAGE and Western Blot analysis. The results showed that HA protein was a 55ku protein with immunogenicity. The purified HA protein was used to establish the indirect ELISA for detection of the antibodies against H1 subtype of SIV. The assay has excellent specificity, high sensitivity and excellent reproducibility. The positive rate was about 20.5% coming from 2 584 clinical serums in 2006.

关 键 词：猪流感病毒 H1N1亚型 血凝素基因 间接ELISA 

分 类 号：S852.659.5[农业科学—基础兽医学] S858.28[农业科学—兽医学]