基因Ⅳ型禽呼肠孤病毒σC蛋白单克隆抗体的制备及功能鉴定
Preparation and Functional Characterization of Monoclonal Antibodies against σC Protein of Genotype Ⅳ Avian Reovirus
高金慧 1王素艳 2刘芮 2祁小乐 2陈运通 2张艳萍 2崔红玉 2刘永振 2段雨路 2高立 2高玉龙2
作者信息
- 1. 黑龙江八一农垦大学动物科技学院,黑龙江大庆 163000
- 2. 中国农业科学院哈尔滨兽医研究所,动物疫病防控全国重点实验室,黑龙江哈尔滨 150069
- 折叠
摘要
为制备基因Ⅳ型(GC Ⅳ)禽呼肠孤病毒(avian reovirus,ARV)σC蛋白单克隆抗体,构建了重组原核表达质粒pCold-I-ARV-GC Ⅳ-σC,将重组质粒导入感受态细胞BL21(DE3)中进行蛋白诱导表达;随后将纯化后的σC蛋白免疫BALB/c小鼠,通过细胞融合技术制备杂交瘤细胞,利用间接酶联免疫吸附试验(ELISA)筛选获得阳性杂交瘤细胞;以间接免疫荧光(IFA)和蛋白免疫印迹(Western blot)试验鉴定杂交瘤细胞后,将杂交瘤细胞株通过腹腔注射小鼠制备单克隆抗体腹水,以ELISA测定腹水效价.结果显示:成功筛选出一株分泌ARV GC Ⅳ σC蛋白单克隆抗体的阳性杂交瘤细胞株(1H4);制备的单克隆抗体能与ARV GC Ⅳ σC蛋白发生反应,同时与感染不同基因型ARV的鸡肝癌细胞(LMH)均有良好的反应性;该单克隆抗体识别重链为IgG1,其腹水ELISA效价为 1:256 000.结果表明,成功制备ARV GC Ⅳ σC蛋白单克隆抗体,其效价高,反应性良好,为进一步研究σC蛋白功能、ARV致病机理以及建立临床诊断方法奠定了基础.
Abstract
In order to prepare monoclonal antibodies against σC protein of genotype Ⅳ(GC Ⅳ)avian reovirus(ARV),the recombinant prokaryotic expression plasmid pCold-I-ARV-GC Ⅳ-σC was constructed and transformed into the BL21(DE3)competent cells for protein expression;subsequently,the purified σC protein was immunized to BALB/c mice,and hybridoma cells were prepared by cell fusion,and positive ones were screened by indirect enzyme-linked immunosorbent assay(ELISA);the hybridomas,after identification by indirect immunofluorescence(IFA)and Western blot,was intraperitoneally injected into the mice to prepare monoclonal antibody(mAb)ascites whose titer was measured by ELISA.The results showed that a positive hybridoma cell line(1H4)secreting mAb against ARV GC Ⅳ σC protein was successfully screened;the prepared mAb could react with ARV GC Ⅳ σC protein,and well interact with LMH cells infected with different genotypes of ARV;the mAb heavy chain was identified as IgG1,and the ELISA titer of ascites was 1:256 000.In conclusion,mAb against ARV GC Ⅳ σC protein with good titer and reactivity were successfully prepared in this study,laying a foundation for further researches on the functions of ARV GC Ⅳ σC protein,the pathogenesis of ARV and the development of clinical diagnostic methods.
关键词
禽呼肠孤病毒/基因Ⅳ型/σC蛋白/单克隆抗体/功能鉴定Key words
ARV/genotype Ⅳ/σC protein/monoclonal antibody/function identification引用本文复制引用
基金项目
国家重点研发计划项目(2022YFF0710500)
中央级公益性科研院所基本科研业务费专项(1610302022014)
国家肉鸡产业技术体系项目(CARS-41)
出版年
2024
国家科技期刊平台
NSTL
万方数据