猪瘟病毒E2蛋白主要抗原区高效表达及间接ELISA抗体检测方法的建立和应用  

Efficient expression of the major antigen region of the E2 gene of the classical swine fever virus and development of indirect ELISA for antibody detection

作　　者：陈九 潘丽[2] 马中元 崔燕[1] CHEN Jiu;PAN Li;MA Zhongyuan;CUI Yan(Gansu Agricultural University,Lanzhou 730000,China;Lanzhou Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Lanzhou 730000,China)

机构地区：[1]甘肃农业大学,甘肃兰州730000 [2]中国农业科学院兰州兽医研究所,甘肃兰州730000

出　　处：《畜牧与兽医》2020年第6期100-106,共7页Animal Husbandry & Veterinary Medicine

摘　　要：利用大肠杆菌表达猪瘟病毒E2蛋白主要抗原区,建立猪瘟病毒间接ELISA抗体检测方法。依据大肠杆菌密码子偏好性对猪瘟病毒E2基因主要抗原区的稀有密码子进行同义替换,克隆至pET-30a(+),转化大肠杆菌BL21(DE3)构建重组表达菌,并将纯化重组蛋白作为抗原建立猪瘟病毒间接ELISA抗体检测的方法。对重组蛋白进行SDS-PAGE和Western blot检测,结果显示获得分子量为30 ku的重组蛋白,占细菌总蛋白的41%,与猪瘟抗体特异性反应强;间接ELISA重复性检测显示批内变异系数为3.72%,批间变异系数为4.75%;特异性检测显示,与猪圆环病毒、蓝耳病病毒、伪狂犬病病毒、猪细小病毒、猪流行性腹泻病毒和口蹄疫病毒的抗体阳性血清无交叉反应,并且对566份样品的临床检测结果显示与爱德仕猪瘟阻断ELISA试剂盒的阳性符合率为90.80%,阴性符合率为93.29%,总符合率为91.52%。结果表明密码子优化后E2重组蛋白实现高效表达,建立的猪瘟病毒间接ELISA抗体检测方法成本低、准确率高,可进一步开发应用。The major antigen region of the E2 protein of the classical swine fever virus expressed by Escherichia coli was used for indirect ELISA antibody detection of the virus.Synonymous replacement of rare codons in the major antigenic region of the classical swine fever virus E2 gene was carried out according to the codon preference of Escherichia coli,and the nucleotide sequence were cloned onto PET-30 a(+),and the recombinant expression bacterium was constructed by transforming Escherichia coli BL21(DE3).SDS-PAGE and Western-blot showed that the target protein with a molecular weight of 30 ku accounted for 41%of the total bacterial proteins and had specific reaction with the classical swine fever antibody.The purified recombinant protein was used as the antigen condition to establish an indirect ELISA antibody detection method for the classical swine fever virus.Repeated tests showed that the intra-batch coefficient of variation was 3.72%,and the inter-batch coefficient of variation was 4.75%.Specific tests showed that there was no cross reaction with the positive serum of PCV2,PRRSV,PRV,PPV,PEDV and FMDV.566 serum samples were detected in this study compared with the results of the IDEXX block ELISA kit,with a positive coincidence rate of 90.80%a negative coincidence rate of 93.29%,and a total coincidence rate of 91.52%.Our results suggested that the expression of the E2 gene in Eacterium coli might be successfully improved by codon optimization,and the established indirect ELISA method in this study was of low cost high accuracy and had a prospect of application.

关 键 词：猪瘟病毒 E2蛋白 原核表达 密码子优化 间接ELISA 

分 类 号：S852.65[农业科学—基础兽医学]