猪繁殖与呼吸综合征病毒非结构蛋白7特异性抗体体外抑制病毒复制

Antiviral activity of specific polyclonal antibody against porcine reproductive and respiratory syndrome virus nonstructural protein 7 in vitro

为进一步探究猪繁殖与呼吸综合征病毒(porcine reproductive and respiratory syndrome virus,PRRSV)非结构蛋白7(Nsp7)在病毒复制过程中发挥的生物学作用,本研究在获得Nsp7可溶性重组蛋白的基础上,首先制备了抗Nsp7蛋白兔源多克隆抗体。确定抗体的免疫活性后,将其与PRRSV共同接种Marc-145细胞。孵育1h后弃掉混合物,加细胞维持液继续培养48h,应用TaqMan探针Real Time PCR方法对培养物中病毒拷贝数进行检查。结果显示,本研究制备的多克隆抗体可以和大肠杆菌表达的Nsp7α、Nsp7β重组蛋白发生特异性结合,并有效用于病毒感染Marc-145细胞后其Nsp7蛋白分布情况的原位检测;更为重要的是,与正常家兔血清相比,不同稀释倍数的抗Nsp7多克隆抗体均可对病毒的复制产生抑制作用,其中10-3倍稀释血清的抑制率可达88.4%。结果表明,Nsp7特异性抗体可有效用于PRRSV抑制试验的研究;Nsp7作为PRRSV复制酶多聚蛋白成员之一,在病毒复制过程中发挥着重要作用。

Porcine reproductive and respiratory syndrome virus(PRRSV)nonstructural protein 7(Nsp7)recombinant protein was obtained and its polyclonal antibody originated from rabbit was prepared in this study to explore the biological function of PRRSV Nsp7 protein in the process of virus replication.When the immuno-activity of this polyclonal antibody was confirmed,it was inoculated into Marc-145 cells line together with the PRRSV.At 1hpost-incubation,the mixture of virus and antibody was dropped,and new maintenance media was added for another 48 h.The TaqMan probe real time PCR method was used to detect the copy number of the virus in media.The results revealed that the antibody could react to Nsp7αand Nsp7βrecombinant protein expressed in Escherichia coli,and be used to detect the distribution of Nsp7 protein produced by PRRSV.More importantly,the different dilutions of antibodies against Nsp7 protein all inhibited virus replication compared with the control group,and the antibody of 10-3 dilution has an inhibition ratio of 88.4%.In brief,the specific polyclonal antibody against Nsp7 could be effectively used for the PRRSV inhibition research,and this result could indirectly showed that Nsp7 as a member of PRRSV replicase polyprotein may have an important effect in the process of virus replication.