A型口蹄疫病毒型特异性抗原的可溶性原核表达及多克隆抗体的制备  

Soluble expression of the type-specific antigen of FMDV of serotype A and preparation of its polyclonal antibody

作　　者：高闪电[1] 常惠芸[1] 独军政[1] 丛国正[1] 邵军军[1] 林彤[1] 

机构地区：[1]中国农业科学院兰州兽医研究所,国家口蹄疫参考实验室,家畜疫病病原生物学国家重点实验室,农业部畜禽病毒学重点开放实验室,兰州730046

出　　处：《中国人兽共患病学报》2010年第9期821-824,共4页Chinese Journal of Zoonoses

基　　金：国家支撑计划资助项目(No.2006BAD06A14)

摘　　要：目的可溶性表达A型口蹄疫病毒型特异性结构蛋白VP1,制备型特异性的兔抗A型FMDV VP1多克隆抗体。方法以A型口蹄疫病毒结构蛋白VP1基因重组克隆载体pGEM-AVP1为模板,设计表达引物,经PCR扩增获得上游带有OmpT信号肽编码序列的VP1基因编码区,将其克隆至该表达载体pET-30a(+)中,构建可溶性原核表达载体pET-30a-OmpT-AVP1。将该重组质粒转化大肠杆菌工程菌株BL21-(DE3)-plysS,经IPTG诱导表达,以金属离子螯合层析法对可溶性表达的VP1融合蛋白进行纯化,然后用纯化的蛋白免疫新西兰大耳白兔制备其多克隆抗体,以ELISA、Western blot和间接免疫荧光法分别对多克隆抗体进行鉴定。结果 SDS-PAGE电泳分析显示pET-30a-OmpT-AVP1重组菌经诱导后表达一Mr约为33 000的VP1融合蛋白,与预期结果相符。目的蛋白纯化后,在电泳图片上显示较为清晰的单一条带。Western blot分析结果显示,VP1蛋白可与豚鼠抗A型口蹄疫病毒阳性血清反应。用纯化VP1蛋白免疫新西兰大耳白兔制备的多克隆抗体效价达1∶12 800以上,具有较高的型特异性,并且可与同型FMDV细胞培养物中的病毒抗原发生反应。结论制备了具有高亲和性和特异性的兔抗A型FMDV结构蛋白VP1多克隆抗体,为A型FMDV易感动物的抗体筛查及病原鉴定奠定了基础。To express soluble recombinant type-specific antigen of foot-and-mouth disease virus of serotype A and prepare polyclonal antibody that of type specificity,the recombinant cloning vector pGEM-AVP1 was used as template for PCR to get the coding region of VP1,and the coding region of OmpT signal peptide was added to the upstream of VP1,then the fragment was cloned to pET-30a（＋） to get recombinant expression plasmid pET-30a-OmpT-AVP1.The E.coli BL21-（DE3）-plysS was transformed and expressed soluble fused VP1 with the expected molecular weight at 33 000.Purified VP1 was obtained by Ni-NTA His Bind Resin affinity chromatography and could react with the positive sera of guinea pig of the same serotype.New Zealand rabbits were immunized to prepare polyclonal antibody against VP1.Antiserum against VP1 was obtained and polyclonal antibody was characterized by ELISA,Western blot and indirect immunofluorescence assay（IFA）.The polyclonal antibody titer was above 1∶12 800 and type-specific.Moreover,the polyclonal antibody could react well with viral antigen in cell culture.Type-specific antigen of foot-and-mouth disease virus of serotype A and its polyclonal antibody were prepared successfully,which will lay a foundation for further antibody and virus detection.

关 键 词：A型口蹄疫病毒 结构蛋白VP1 原核表达 可溶性 OmpT信号肽 

分 类 号：R392.11[医药卫生—免疫学]