针对不同形式猪繁殖与呼吸综合征病毒GP5重组蛋白的抗体中和活性比较

doi:10.11843/j.issn.0366-6964.2018.06.014

摘要/Abstract

摘要：

GP5蛋白被认为是猪繁殖与呼吸综合征病毒诱导中和抗体的主要蛋白，为探讨GP5蛋白诱导免疫保护的能力，本研究用杆状病毒/昆虫表达系统分别表达了全长和截短的GP5蛋白，同时用大肠杆菌表达系统表达了截短的GP5蛋白。三种重组蛋白经纯化后，免疫小鼠制备抗血清，以PRRSV全病毒作为ELISA包被抗原检测抗血清时，原核表达的截短GP5蛋白抗血清效价为1∶200，真核表达的全长GP5蛋白抗血清效价为1∶800，真核表达的截短GP5蛋白抗血清效价为1∶1 600。将获得的三种抗血清分别在猪肺泡巨噬细胞（porcine alveolar macrophages，PAMs）进行PRRSV同源（PRRSV VR2332）和异源（PRRSV HeN-3）毒株的血清学中和试验，并通过实时定量PCR方法检测各组中PRRSV mRNA的绝对拷贝数，以抗血清的病毒阻断率≥70%的最大血清稀释度作为该血清的中和效价。结果显示：1）三种重组蛋白的抗血清对两种PRRSV毒株的中和效价均<1∶2，不具有中和活性，部分稀释滴度的血清还增强了病毒的感染；2）抗PRRSV全病毒对照阳性血清对同源毒株的中和效价为1∶8，具有中和活性；对异源毒株的中和效价<1∶2，不具有中和活性，同时还产生了ADE增强作用。上述研究结果提示，GP5的主要中和表位可能更多是构象表位，PRRSV全病毒中主要诱导中和抗体产生的蛋白可能并不完全在GP5蛋白上；PRRSV全病毒阳性抗血清虽然能够提供一定的免疫保护，但这种免疫保护只发生在同源毒株之间，对异源毒株并没有效果，反而会介导ADE效应发生。

Abstract:

GP5 protein of porcine reproductive and respiratory virus (PRRSV) is considered to be the main protein that inducing production of neutralizing antibodies. In order to investigate the ability of the protective immunity induced by the GP5 protein, the full-length GP5 and the truncated GP5 lacking of the signal peptide (aa 1-31) and transmembrane region (aa 66-125) were expressed using baculovirus and prokaryotic expression systems. Additionally, the truncated GP5 lacking of the signal peptide(aa 1-31) and transmembrane region (aa 66-125) were expressed using E. coli expression systems. Three anti-sera (ELISA assay antibody titre:800, 1 600, and 200 respectively) specific for each recombinant protein were derived from mice immunized with three recombinant proteins with Freund's Incomplete Adjuvant. PAM cells used in neutralization assay were obtained from PRRSV-negative healthy pigs by bronchoalveolar lavage after necropsy. PRRSV HeN-3 (the heterologous strain) and VR2332 (the homologous strain) strains were isolated in Marc-145 cells. The real-time quantitative PCR was used to detect PRRSV RNA levels in infected PAM cells. It was considered positive that virus blocking rate was higher than 70% which had been established in our laboratory. Results were as follows:1) The results showed that three anti-sera specific for three recombinant proteins did not have neutralizing activities against two strains of PRRSV (titers were < 1:2). 2) The anti-sera of PRRSV VR2332 had neutralizing activity against PRRSV VR2332 strain (titers were 1:8), and hadn't neutralizing activity against PRRSV HeN-3 strain but rather enhanced infection. Our current work showed the main neutralizing epitopes of GP5 were probably conformational epitopes and the neutralizing epitopes of PRRSV were not fully presented in the GP5 protein. The positive anti-sera of PRRSV could provide immune protection, but the immune protection occurs only in the same strain. It would mediate the ADE effect on heterologous strains on the contrary.

中图分类号:

冯延, 郭嘉, 许瑞勤, 马思续, 魏凤灵, 张留君, 杨国宇, 夏平安, 张改平. 针对不同形式猪繁殖与呼吸综合征病毒GP5重组蛋白的抗体中和活性比较[J]. 畜牧兽医学报, 2018, 49(6): 1222-1230.

FENG Yan, GUO Jia, XU Rui-qin, MA Si-xu, WEI Feng-ling, ZHANG Liu-jun, YANG Guo-yu, XIA Ping-an, ZHANG Gai-ping. Comparison of Neutralizing Antibodies Induced by PRRSV GP5 Recombinant Proteins in Different Forms of Truncation[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2018, 49(6): 1222-1230.

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