摘要
【目的】构建猪繁殖与呼吸综合征病毒(PRRSV)Nsp10蛋白原核表达系统,制备多克隆抗体,系统评价其免疫原性,为基于Nsp10蛋白的血清学诊断方法建立与疫苗研发提供理论依据。【方法】利用ExPASy、IEDB和SWISS-MODEL分别预测Nsp10蛋白的亲/疏水性、抗原表位和三级结构。构建重组质粒pET-28a-Nsp10,转化大肠杆菌BL21(DE3)感受态细胞进行诱导表达,优化诱导温度、IPTG浓度和诱导时间。采用His-Bind镍离子亲和层析柱纯化重组Nsp10蛋白(rNsp10),利用SDS-PAGE与Western blotting检测纯化效果。使用纯化后的rNsp10蛋白免疫C57BL/6J小鼠,利用间接ELISA法测定抗体效价与亚型。采用实时荧光定量PCR检测脾细胞中免疫应答相关基因的相对表达量,利用Western blotting与间接免疫荧光检测抗体反应原性。【结果】Nsp10蛋白为亲水性蛋白,存在19个B细胞抗原表位,具备作为多克隆抗体制备靶抗原的潜力。利用原核表达系统成功构建重组质粒pET-28a-Nsp10,在18~37℃条件下使用1.5 mmol/L IPTG诱导8 h可实现rNsp10蛋白的高效表达,且其主要以包涵体形式存在,纯化后产量达15 mg/L。以rNsp10蛋白免疫C57BL/6J小鼠可诱发强烈的体液免疫应答,抗体效价最高达1∶409600,且能在免疫后期维持较高水平。抗体亚型与免疫应答检测结果显示,rNsp10蛋白免疫不仅能诱导以IgG1为主的体液免疫,还能极显著升高Th2型体液免疫相关基因IL-4和IL-10的相对表达量与Th1型细胞免疫相关基因IL-6、IL-1β、IFN-γ、TNF-α和IL-12的相对表达量(P<0.01)。rNsp10多克隆抗体反应原性检测结果显示,鼠源抗rNsp10多克隆抗体能特异性识别PRRSV在感染过程中表达的天然蛋白Nsp10,具有良好的反应原性。【结论】PRRSV Nsp10蛋白为亲水性蛋白,存在19个B细胞抗原表位,适合采用原核系统进行表达。rNsp10蛋白具有良好的免疫原性,能同时激活以IgG1为主的体液免疫应答与以IFN-γ和IL-12等基因为特征的细胞免疫。rNsp10鼠源多克隆抗体能特异性识别PRRSV在感染过程中表达的天然蛋白Nsp10。
【Objective】This study aimed to construct a prokaryotic expression system for the Nsp10 protein of porcine reproductive and respiratory syndrome virus(PRRSV),prepare polyclonal antibodies,and evaluate their antigenicity,thereby providing a theoretical basis for establishment of serological diagnosis methods and vaccine research and development based on Nsp10 protein.【Method】The hydrophilicity/hydrophobicity,antigenic epitopes,and tertiary structure of the Nsp10 protein were analyzed respectively using ExPASy,IEDB,and SWISS-MODEL.The recombinant plasmid pET-28a-Nsp10 was constructed and transformed into Escherichia coli BL21(DE3)competent cells for induced expression.The induction temperature,IPTG concentration,and induction time were optimized.The recombinant Nsp10 protein(rNsp10)was purified using a His-Bind nickel ion affinity column,and the purification effects were assessed by SDS-PAGE and Western blotting.The purified rNsp10 was used to immunize C57BL/6J mice,and antibody titers and isotypes were determined by the methed of indirect ELISA.The relative expressions of immune response-related genes in splenocytes were measured using real-time fluorescence quantitative PCR,while antibody reactogenicity was evaluated by Western blotting and indirect immunofluorescence.【Result】The Nsp10 protein was hydrophilic and contained 19 B-cell antigenic epitopes,suggesting its potential as a target antigen for polyclonal antibody preparation.The recombinant plasmid pET-28a-Nsp10 was constructed in a prokaryotic expression system.The high-efficiency expression of rNsp10 protein was achieved under induction with 1.5 mmol/L IPTG for 8 h at 18 to 37℃,with the protein mainly existing as inclusion bodies and purified yield of 15 mg/L.Immunization of C57BL/6J mice with rNsp10 elicited a strong humoral immune response,with a maximum antibody titer of 1∶409600,which remained at high levels during the later stages of immunization.Antibody isotype and immune response tests revealed that rNsp10 immunization not only induced a humoral immune response dominated by IgG1,but also significantly up-regulated the relative expressions of genes related to Th2 humoral immune response IL-4 and IL-10,as well as genes related to Th1 cellular immune response IL-6,IL-1β,IFN-γ,TNF-α,and IL-12(P<0.01).Reactogenicity test of the rNsp10 polyclonal antibody showed that mouse-derived anti-rNsp10 polyclonal antibodies could specifically recognize native Nsp10 protein expressed during PRRSV infection,demonstrating good reactogenicity.【Conclusion】PRRSV Nsp10 is a hydrophilic protein containing 19 B-cell epitopes and is suitable for expression in a prokaryotic system.The rNsp10 protein exhibits strong immunogenicity,simultaneously capable of activating a humoral immune response dominated by IgG1 and a cellular immune response characterized by genes such as IFN-γand IL-12.rNsp10 mouse-derived polyclonal antibodies specifically recognize native Nsp10 protein expressed during PRRSV infection.
作者
王辰怡
傅威
李若铭
陈佳佳
李名志
夏晓锦
翁雅萍
孔令保
彭棋
王婷
WANG Chen-yi;FU Wei;LI Ruo-ming;CHEN Jia-jia;LI Ming-zhi;XIA Xiao-jin;WENG Ya-ping;KONG Ling-bao;PENG Qi;WANG Ting(College of Bioscience and Engineering,Jiangxi Agricultural University,Nanchang,Jiangxi 330045,China;Nanchang Key Laboratory of Animal Virus and Genetic Engineering,Nanchang,Jiangxi 330045,China)
出处
《南方农业学报》
北大核心
2025年第10期3241-3249,共9页
Journal of Southern Agriculture
基金
国家自然科学基金项目(31960699)
江西省自然科学基金项目(20224BAB215001,20242BAB23068)。
关键词
猪繁殖与呼吸综合征病毒
Nsp10蛋白
原核表达
多克隆抗体
免疫原性
porcine reproductive and respiratory syndrome virus
Nsp10 protein
prokaryotic expression
polyclonal antibody
immunogenicity
