猪繁殖与呼吸综合征病毒NJ-a株ORF6基因的表达及特异性抗体的制备  

Expression for the ORF_6 gene of PRRSV NJ-a strain in Escherichia coli and its preparation of the specific polyclonal antiserum

作　　者：郑其升[1] 杨瑞锋 毕志香 李鹏[1] 于春梅[1] 曹瑞兵[1] 陈溥言[1] 

机构地区：[1]南京农业大学农业部动物疫病诊断与免疫重点开放实验室,江苏南京210095 [2]山东省畜牧兽医职业技术学院,山东潍坊262101

出　　处：《南京农业大学学报》2008年第2期154-158,共5页Journal of Nanjing Agricultural University

摘　　要：根据GenBank公布的猪繁殖与呼吸综合征病毒(porcine reproductive and respiratory syndrome virus,PRRSV)VR2332株的核酸序列设计并合成1对特异性引物,用RT-PCR方法扩增PRRSV NJ-a株ORF6基因,将其连接入pMD18-T载体,经测序后克隆入原核表达载体pET-32a(+)上,构建原核表达载体pET-ORF6。阳性质粒转化感受态大肠杆菌BL21(DE3),经异醛-β-D-硫代半乳糖苷(IPTG)诱导,PRRSVORF6基因获得表达。以纯化的重组蛋白为抗原免疫家兔3次,获得抗PRRSV NJ-a株M蛋白的特异抗体。经Western-blotting证明,该多克隆抗体既能与原核表达的重组M蛋白发生反应,又能与PRRSV发生特异性反应;间接免疫荧光结果表明,获得的多克隆抗体与表达PRRSV M蛋白的293T细胞及感染PRRSV的Marc-145细胞发生反应。兔抗PRRSV NJ-a株M蛋白特异性抗体的获得,为进一步研究PRRSV M蛋白的表达和功能奠定了基础。With a pair of specific primers designed according to the relevant sequence of porcine reproductive and respiratory syndrome virus （PRRSV） VR2332 strain （ accession NO. AF030244） , the ORF6 gene of PRRSV NJ-a strain isolated in our laboratory from Nanjing was amplified with RT-PCR method. The PCR product was cloned into pMD18-T vector and then sequenced. Fol- lowing that, it was directly cloned into plasmid pET-32a （ ＋ ） to obtain a prokaryotic expression vector pET-ORF6. The positive plasmid was transformed into the host cell E. coli BL21 （ DE3 ） and the ORF6 gene was successfully expressed with the induction of 1.0 mmol · L^-1 isopropy β-D-1-thiogalactopyranoside （IPTG）. The specific polyclonal antiserum was obtained from rabbit immunized with the purified recombinant fusion protein. Western blotting was applied to prove that the antiserum can react with both recombinant fusion M protein and PRRSV. Also, IFA test demonstrated that this polyclonal antiserum could react with the 293T cells which can express PRRSV M protein and the Marc-145 cells infected with PRRSV. Conclusions： the rabbit anti-PRRSV M protein polyclonal antiserum obtained in this study laid a foundation for further functional research and detection for the expression of M protein in eukaryotic system.

关 键 词：猪繁殖与呼吸综合征病毒NJ-a株 ORF6基因 原核表达 抗体制备 

分 类 号：S852.43[农业科学—基础兽医学]