口蹄疫病毒3D聚合酶多克隆抗体的制备与特性分析  

Preparation and identification of the polyclonal antibody against the 3D polymerase of foot and mouth disease virus

作　　者：张昱[1] 王永录[1] 张永光[1] 方玉珍[1] 潘丽[1] 蒋守田[1] 吕建亮[1] 刘力宽[1] 张中旺[1] 张淑刚[1] 李正丰[1] 杜进鑫[1] 

机构地区：[1]中国农业科学院兰州兽医研究所,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1期6-9,共4页Chinese Journal of Zoonoses

基　　金：国家支撑计划项目(2006BAD06A06)资助

摘　　要：目的表达口蹄疫病毒3D聚合酶,并制备兔抗3D多克隆抗体。方法采用PCR方法扩增口蹄疫病毒(FM-DV)3D聚合酶基因,经BamHⅠ和NdeⅠ双酶切后插入pET-30a(+)原核表达载体,构建的pET-3D重组表达质粒转化大肠杆菌BL21(DE3)菌株,经IPTG诱导表达,SDS-PAGE鉴定目的蛋白的表达并提取包涵体,纯化目的蛋白。然后用纯化蛋白免疫新西兰大耳白兔制备多克隆抗体,以Western blot鉴定抗体特异性,并以间接ELISA方法测定其效价。结果SDS-PAGE电泳分析显示,表达的目的蛋白约为46ku,与预期结果相符,制备的多克隆抗体效价达1∶8000以上,且具有较高的特异性。结论制备了具有高亲和性和特异性的3D聚合酶多克隆抗体,为3D聚合酶的生物学功能和抗原表位研究奠定了基础。To prepare the polyclonal antibody against the 3D polymerase of foot and mouth disease virus（FMDV）,the 3D polymerase gene of this virus was amplified by PCR and doubly digested with BamH I and Nde I.Then,it were cloned into expression vector pET-30a（＋）to obtain the recombinant plasmid pET-3D and this plasmid was transformed to E.coli BL21（DE3）with induction by IPTG.The target protein was identified and purified with SDS＋PAGE,and the inclusion bodies were extracted.The purified target protein was used as antigen to immunize New Zealand rabbits to prepare the polyclonal antibody against 3D polymerase of FMDV,which was then characterized by indirect ELISA and Western blotting.As demonstrated by SDS-PAGE,the target protein with a molecular weight at 46 ku was expressed.The polyclonal antibody showed high affinity and obvious specificity and its titer was above 1∶8 000.This polyclonal antibody may lay a foundation for the further studies on the biological functions and epitopes of the 3D polymerase of FMDV.

关 键 词：口蹄疫病毒 3D聚合酶 多克隆抗体 

分 类 号：S852.65[农业科学—基础兽医学]