猪繁殖与呼吸综合征病毒nsp4抗体制备与鉴定

摘要

To obtain specific antibodies against nsp4 protein of porcine reproductive and respiratory syndrome virus (PRRSV),nsp4 gene was amplified by RT-PCR and cloned into pET-28a(+) vector,designated pET28a-nsp4,pET28a-nsp4 was transformed into Escherichia coli Trasseta (DE3) cells and expressed after induction of IPTG.SDS-PAGE analysis showed that the recombinant protein was expressed in soluble form with the molecular weight of 26 kDa.The soluble fusion protein in the supernatant was purified using Ni+-NTA affinity chromatography.New Zealand rabbits were immunized by the purified nsp4 and anti-sera against nsp4 were obtained.The titer of polyclonal antibodies was about 106 and showed good specificity and sensitivity in the immunofluorescence assay and Western blotting analysis.The polyclonal antibodies also recognized native nsp4 form PRRSV infected Marc-145 cells,providing a useful tool in PRRSV replication mechanism study.%为了获得猪繁殖与呼吸综合征病毒(PRRSV) nsp4的抗体,根据HP-PRRSV TA-12株(GenBank Accession No.HQ416720)的nsp4基因序列,设计并合成一对引物.用RT-PCR扩增后克隆到原核表达载体pET-28a(+)中,构建重组质粒pET28a-nsp4,转化至Trasseta(DE3),经IPTG诱导重组蛋白获得了高效可溶性表达,大小约为26 kDa.经镍离子亲和柱(Ni+-NTA)纯化获得了高纯度重组蛋白,将纯化的nsp4蛋白免疫新西兰大白兔制备了多克隆抗体.ELISA检测抗体效价可达106,Western blotting和IFA检测结果表明所制备的多克隆抗体具有良好的免疫反应特异性,能够识别PRRSV感染宿主细胞中的nsp4蛋白.本研究成功制备了针对nsp4的多克隆抗体,为进一步研究nsp4的功能及PRRSV致病机制奠定了基础.