猪繁殖与呼吸综合征病毒GP5重组蛋白对外周血单个核细胞功能的影响

Effect of recombinant GP5 protein of Porcine reproductive and respiratory syndrome virus on peripheral blood mononuclear cells

为了研究猪繁殖与呼吸综合征病毒(PRRSV)GP5重组蛋白(rGP5)对猪外周血单个核细胞(PBMC)功能的影响,本研究选取PRRSV-PC疫苗株中的ORF5基因片段,对其进行原核表达并纯化,结果显示rGP5以可溶性和包涵体两种形式表达,分子质量约为30 ku;将该纯化蛋白免疫大耳白兔,按常规方法制备抗血清并进行酶联免疫吸附试验(ELISA),结果显示,纯化后的rGP5可以与PRRSV阳性血清特异性结合;制备的兔抗rGP5多克隆抗体效价高达1.64 000,可用于后续试验。无菌采集PRRSV阴性猪外周血并分离PBMC,加入终浓度为10μg/mL的rGP5体外培养后,采用间接免疫荧光试验(IFA)检测重组蛋白与PBMC的结合,结果显示rGP5与PBMC共培养1 h后,PBMC出现红色荧光,表明r GP5可以结合PBMC。进一步采用不同浓度的rGP5体外刺激猪PBMC,利用ELISA方法检测淋巴细胞中IL-10、IFN-γ、TGF-β1及TNF-α的分泌水平,同时采用CCK-8细胞增殖检测试剂盒测定rGP5对PBMC增殖的影响,以及不同浓度的rGP5刺激PBMC分泌NO的情况,结果显示,不同浓度的rGP5均显著促进PBMC细胞因子IL-10、IFN-γ和TNF-α的分泌(p<0.05);同时rGP5能够促进PBMC增殖(p<0.05),也能增加NO分泌(p<0.05),上述结果表明GP5是PRRSV重要的抗原,发挥着重要的免疫作用。本实验首次研究了rGP5对猪PBMC免疫功能的影响,揭示PRRSV影响宿主免疫反应的具体机制,有助于阐明PRRSV与宿主细胞相互作用的分子基础,为进一步研究其免疫保护性提供了实验依据。

To study the effect of recombinant GP5 protein of porcine reproductive and respiratory syndrome virus(PRRSV) on the function of porcine peripheral blood mononuclear cells(PBMC), the ORF5 gene fragment of PRRSV-PC vaccine strain was expressed in prokaryotic system and purified as recombinant protein r GP5. The results showed that r GP5 was expressed as soluble and inclusion bodies with a molecular weight of about 30 ku in E. coli cells. The purified protein was used to immunize the white big-ear rabbits and the antiserum was collected and tested by ELISA. The results showed that the purified r GP5 could specifically bind with serum against PRRSV. The titer of rabbit anti r GP5 polyclonal antibody was as high as 1:64000, which can be used in follow-up test. The peripheral blood mononuclear cells(PBMCs) were isolated from the blood of PRRSV free pig and cultured with r GP5 of 10μg/m L in vitro. The recombinant protein binding to PBMCs was analyzed by indirect immunofluorescence assay(IFA). The r GP5 stained with red could bind to PBMC after co-culture for 1 hour in IFA. The secretion of IL-10, IFN-γ, TGF-β1 and TNF-α in porcine PBMC was detected by ELISAs after being stimulated with different concentrations of r GP5, and the effect of r GP5 on PBMC proliferation was detected by CCK-8 cell proliferation assay kit. The results showed that the amounts of IL-10,IFN-γ and TNF-αwere increased significantly(p<0.05) by the stimulation of r GP5 in PBMC. The r GP5 enhanced the proliferation of PBMC(p<0.05) and NO secretion(p<0.05), indicating that r GP5 is an important antigen of PRRSV and plays an important role in immune fuction. This study investigated the effect of the recombinant r GP5 on the immune function of PBMCs and will contribute to elucidate the molecular mechanism of PRRSV interaction with host cells, revealed the specific mechanism of PRRS affecting host immune response and inducing immune escape, and provided experimental evidence for the further study of the interaction of PRRSV with host.