伪狂犬病病毒UL36蛋白单克隆抗体的制备和鉴定
Preparation and identification of monoclonal antibody against pseudorabies virus UL36 protein
陈玲超 1王兴娅 2李俊波 3李佳佳 4王晨 1李松楠 1王安铖 2孟鑫 3童武 1孔宁 1于海 1单同领 1童光志 1郑浩1
作者信息
- 1. 中国农业科学院上海兽医研究所,上海 200241
- 2. 中国农业科学院上海兽医研究所,上海 200241;安徽农业大学生命科学院,安徽合肥 230036
- 3. 中国农业科学院上海兽医研究所,上海 200241;东北林业大学野生动物学院,黑龙江哈尔滨 150040
- 4. 中国农业科学院上海兽医研究所,上海 200241;安徽农业大学动物科技学院,安徽合肥 230036
- 折叠
摘要
伪狂犬病病毒UL36基因编码的皮层蛋白VP1/2在病毒粒子组装过程中发挥重要作用.本试验以UL36基因为研究对象,将截短的UL36基因克隆入载体pColdI,构建了重组原核表达质粒pColdI-UL36.经诱导表达,将包涵体形式表达的重组蛋白经过变性、亲和层析及复性后获得了纯度较高的蛋白.将纯化的蛋白免疫BALB/c小鼠,通过细胞融合和间接ELISA方法筛选出1株能稳定分泌针对UL36蛋白的单克隆抗体杂交瘤细胞株,被命名为UL36-6,抗体亚型鉴定结果为IgG1和κ,制备的腹水抗体中和效价及纯度高,Western-blot和间接免疫荧光试验结果表明单克隆抗体具有很好的特异性.综上所述,本研究成功制备出1株针对PRV UL36蛋白的单克隆抗体,为深入研究UL36蛋白的生物学功能奠定了基础.
Abstract
VP1/2,a tegument protein encoded by the pseudorabies virus UL36 gene,plays an important role in the assembly of virions.In this study,UL36 gene was mainly used as the research object,the truncated UL36 gene was cloned into pCold I vector to construct the recombination prokaryotic expression plasmid pCold I-UL36.After induced expression,the recombinant protein expressed in inclusion body form was denatured,affinity chromatography and renaturation to obtain high purity protein.Then,BALB/c mice were immunized with purified protein.A hybridoma cell strain named UL36-6,stably secreting antibodies against UL36 was obtained by cell fusion and indirect ELISA screening.The identification of antibody subtype showed that it was IgG1 and κ.The ascites antibody had high neutralization titer and purity.The Western-blot and indirect immunofluorescence test results showed that the monoclonal antibody had good specificity.In conclusion,we successfully prepared a monoclonal antibody against PRV UL36 protein in the present study which will lay a foundation for further study of the biological function of UL36 protein.
关键词
伪狂犬病病毒/UL36基因/VP1/2/单克隆抗体Key words
pseudorabies virus/UL36gene/VP1/2/monoclonal antibody引用本文复制引用
基金项目
上海市科技创新行动计划(20392002500)
出版年
2024
国家科技期刊平台
NSTL
万方数据