原核高效表达猪繁殖与呼吸综合征病毒核衣壳蛋白的特性及应用  

Characterization and application of nucleocapsid protein of porcine reproductive and respiratory syndrome virus expressed in E.coli

作　　者：吴国平[1] 郭川[1] 曹敏杰[2] 刘冰心[2] 梁银龙[2] 苏文金[2] 

机构地区：[1]厦门大学生命科学院 [2]集美大学生物工程学院,福建厦门361021

出　　处：《中国兽医学报》2007年第2期150-154,共5页Chinese Journal of Veterinary Science

基　　金：福建省科技攻关计划重点资助项目(2003N083);厦门市科技攻关计划重点资助项目(3502Z20031054)

摘　　要：从已构建的PRRSV ORF7重组质粒pUCm-T-ORF7中用PCR扩增ORF7基因亚克隆至表达载体pGEX-4T-3,构建重组表达质粒pGEX-4T-3-ORF7并转化大肠杆菌。经SDS-PAGE及Western blotting鉴定,成功表达了谷胱苷肽转移酶(GST)融合的核衣壳蛋白(N),重组N蛋白表达量约为菌体总蛋白的35%,主要以可溶的形式存在,且能形成同源二聚体。重组N蛋白经谷胱苷肽凝胶(glutathione sepharose 4B)亲和层析后得到高度纯化,并将该蛋白作为抗原建立了间接ELISA检测方法。利用该方法对某猪场76份猪血清进行检测并将结果与IDEXX公司ELISA试剂盒检测结果作比较,2种方法的总符合率达93.4%,检测结果之间差异不显著(P>0.05)。结果表明大肠杆菌表达的重组GST融合N蛋白具有良好的抗原性,因而有望利用该重组蛋白开发为试剂盒应用于临床PRRSV抗体的检测。Porcine reproductive and respiratory syndrome virus(PRRSV) FJ-1 was a newly identified virus isolate in Fujian province.The ORF7 gene of FJ-1 was amplified by RT-PCR and cloned into vector pUCm-T,then subcloned into expression vector of pGEX-4T-3.The recombinant GST-tagged nucleocapsid protein(rN) was expressed in E.coli and the molecular weight was approximately 38 000 as identified by SDS-PAGE and Western blotting.Expression level of the rN protein was approximately 35% of the total bacterial protein and mostly soluble.The rN protein was purified to homogeneity using GST affinity chromatography.Analysis of the rN protein under nonreducing conditions revealed that similar to native protein,the rN protein also possesses homo-dimerization property.An indirect enzyme-linked immunosorbent assay(ELISA) for detecting PRRSV antibody was developed using the purified rN protein as antigen.76 serum samples were detected by the method and the result was compared with that using IDEXX PRRS HerdChek ELISA kit.An identity of 93.4% was revealed between the two ELISA kits and no significant difference(P>0.05) was detected.The data indicate the rN protein has the potential usefulness for detection of the PRRSV antibodies.

关 键 词：猪繁殖与呼吸综合征病毒 核衣壳蛋白 同源二聚体 间接ELISA 

分 类 号：S852.659[农业科学—基础兽医学]