猪伪狂犬病毒变异株gB基因主要抗原表位区的鉴定OA
Identification of Major Epitope Regions in the gB Gene of Variant Strains of Pseudorabies Virus in Pigs
采用分段表达策略分析猪伪狂犬病毒(PRV)变异株 gB蛋白的抗原表位区域.针对 PRV 变异株的 gB 基因设计 5 对特异性引物,成功扩增出包含不同抗原表位特征区的 5 个基因片段(gB-1 至 gB-5).将各片段定向克隆至 pET-28a表达载体构建重组质粒,经双酶切和测序验证后,转化至大肠杆菌 BL21(DE3)感受态细胞,经 IPTG 诱导重组蛋白表达.SDS-PAGE电泳结果显示,gB-3(16 kDa)、gB-4(30 kDa)和 gB-5(30 kDa)片段在大肠杆菌中实现高效表达.Western Blot分析结果显示,3 种重组融合蛋白均能与抗 His标签蛋白的单克隆抗体发生特异性反应,同时与收集自河南省不同地区规模化猪场的 PRV阳性猪血清发生特异性免疫反应,其中,与重组蛋白 gB-5 的反应活性显著高于 gB-3 和 gB-4,表明 gB-5 蛋白有效保留了 PRV天然 gB蛋白的关键抗原表位.gB-5 蛋白位于 PRV gB蛋白的第 543～743 位氨基酸区域,在不同 PRV 毒株中高度保守.成功筛选出具有良好抗原活性的 PRV gB基因重组蛋白,为后续建立 PRV 血清学诊断方法提供了重要依据和候选抗原.
In this study,a segmented expression strategy was implemented to analyze the antigenic epitope regions of glycoprotein B(gB)in pseudorabies virus(PRV).Five pairs of specific primers were designed for the gB gene of PRV variant strains,and five gene fragments(gB-1 to gB-5)containing characteristic regions of different antigenic epitopes were successfully amplified.Each fragment was directly cloned into the pET-28a ex-pression vector to construct recombinant plasmids.After identification by double enzyme digestion and sequen-cing,the recombinant plasmids were transformed into BL21(DE3)competent cells,and the recombinant pro-teins were expressed through the IPTG induction system.SDS-PAGE analysis show that the gB-3(16 kDa),gB-4(30 kDa),and gB-5(30 kDa)fragments were highly expressed in Escherichia coli.The results of Western Blot analysis show that all three recombinant fusion proteins have specific reactions with monoclonal antibodies against His-tagged proteins.In addition,the proteins had specific immune reactions with PRV-positive pig sera collected from large-scale pig farms in different areas of Henan Province.Among them,The reactivity with the recombinant protein gB-5 is higher than that with gB-3 and gB-4,indicating that the gB-5 protein effectively retained the key antigenic epitopes of the natural gB protein of PRV.In addition,the gB-5 protein is located at positions 543～743 amino acid region of PRV gB protein and is relatively conserved among different PRV strains.In this study,the recombinant protein of the PRV gB gene with good antigenic activity was successfully screened out,providing an important basis and candidate antigen for the subsequent establishment of serological diagnostic methods for PRV.
ZHAI Hongyue;WANG Ling;SONG Jiajing;WANG Jiabao;CAO Jiajia;LI Dandan;LENG Chaoliang
@@@a.School of Life Science||b.Henan Key Laboratory of Insect Biology in Funiu Mountain||c.Henan Provincial Engineering and Technology Center of Animal Disease Diagnosis and Integrated Control,Nanyang Normal University,Nanyang Henan 473061,China@@@a.School of Life Science||b.Henan Key Laboratory of Insect Biology in Funiu Mountain||c.Henan Provincial Engineering and Technology Center of Animal Disease Diagnosis and Integrated Control,Nanyang Normal University,Nanyang Henan 473061,China@@@a.School of Life Science||b.Henan Key Laboratory of Insect Biology in Funiu Mountain||c.Henan Provincial Engineering and Technology Center of Animal Disease Diagnosis and Integrated Control,Nanyang Normal University,Nanyang Henan 473061,China@@@a.School of Life Science||b.Henan Key Laboratory of Insect Biology in Funiu Mountain||c.Henan Provincial Engineering and Technology Center of Animal Disease Diagnosis and Integrated Control,Nanyang Normal University,Nanyang Henan 473061,China@@@a.School of Life Science||b.Henan Key Laboratory of Insect Biology in Funiu Mountain||c.Henan Provincial Engineering and Technology Center of Animal Disease Diagnosis and Integrated Control,Nanyang Normal University,Nanyang Henan 473061,China@@@a.School of Life Science||b.Henan Key Laboratory of Insect Biology in Funiu Mountain||c.Henan Provincial Engineering and Technology Center of Animal Disease Diagnosis and Integrated Control,Nanyang Normal University,Nanyang Henan 473061,China@@@a.School of Life Science||b.Henan Key Laboratory of Insect Biology in Funiu Mountain||c.Henan Provincial Engineering and Technology Center of Animal Disease Diagnosis and Integrated Control,Nanyang Normal University,Nanyang Henan 473061,China
农业科技
PRV变异株gB基因原核表达抗原表位分析
PRV variant strainsgB geneprokaryotic expressionantigenic epitope analysis
《南阳师范学院学报》 2026 (1)
47-52,6
河南省科技攻关项目"基于M蛋白表位缺失的猪繁殖与呼吸综合征病毒标记疫苗株研制"(252102111005).
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