猪流感病毒血凝素基因原核表达及其在间接ELISA中的初步应用  

Prokaryotic expression of hemagglutinin gene of swine influenza virus and its primary application in indirect ELISA

作　　者：陈艳[1] 乔传玲[1] 丁选亚[1] 杨焕良[1] 辛晓光[1] 韩庆功[1] 陈化兰[1] 

机构地区：[1]中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室农业部动物流感重点开放实验室,黑龙江哈尔滨150001

出　　处：《畜牧与兽医》2008年第11期10-13,共4页Animal Husbandry & Veterinary Medicine

基　　金：国家科技攻关计划项目(2004BA519A55)

摘　　要：利用RT-PCR方法扩增猪流感病毒A/Swine/Henan/11/2005(H1N1)血凝素(HA)基因,克隆于pMD18-T载体进行测序。以pMD18-HA为模板、利用带酶切位点的引物再次扩增HA基因的开放阅读框(ORF),克隆到表达载体pET32a中,经双酶切、测序及PCR鉴定得到阳性重组表达质粒pET-HA,将质粒转化到表达宿主菌BL21(DE3)中,经终浓度为1 mmol/L的IPTG诱导,SDS-PAGE结果显示,HA蛋白获得了高效表达,经Western-blot检测证实表达产物具有良好的免疫学活性,在间接ELISA中的初步应用表明具有良好的抗原反应性。本研究为以重组HA蛋白为抗原建立H1亚型猪流感抗体的ELISA检测方法奠定了基础。Hemagglutinin （HA） gene of A/Swine/Henan/11/2005 （H1NI）, cloned into pMD18-T vector, was sequenced. Primers with the digestion sites of SacI and Hind/Ⅲ were designed based on the conserved regions of both the 5＇-end and Yend of HA gene. The PCR product of HA gene was cloned into pET32a. The recombinant plasmid was confirmed by PCR and restriction enzyme analysis, then transferred into BE21 （DE3） and induced by lmM IPTG. The result of SDS-PAGE indicated that HA protein was expressed effectively. The immune activity was confirmed by Western-blot. The results of indirect ELISA demonstrated that the recombinant protein has a good antigenic reactivity. This study lay a foundation for detection of the antibody against H1NI subtype of swine influenza virus using recombinant HA protein as antigen.

关 键 词：猪流感病毒 HA基因 原核表达 

分 类 号：S852.6[农业科学—基础兽医学]