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 againstσC Protein of Genotype IV Avian Reovirus

作　　者：高金慧 王素艳 刘芮 祁小乐[2] 陈运通 张艳萍[2] 崔红玉[2] 刘永振 段雨路 高立[2] 高玉龙[2] Gao Jinhui;Wang Suyan;Liu Rui;Qi Xiaole;Chen Yuntong;Zhang Yanping;Cui Hongyu;Liu Yongzhen;Duan Yulu;Gao Li;Gao Yulong(College of Animal Science and Technology,Bayi Agricultural University,Daqing 163000,Heilongjiang,China;National Key Laboratory of Animal Disease Prevention and Control,Harbin Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Harbin 150069,Heilongjiang,China)

机构地区：[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

出　　处：《中国动物检疫》2024年第8期98-104,共7页China Animal Health Inspection

基　　金：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)。

摘　　要：为制备基因Ⅳ型(GCⅣ)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-GCⅣ-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 GCⅣσC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 GCⅣσC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1:256000。结果表明,成功制备ARV GCⅣσC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。In order to prepare monoclonal antibodies againstσC 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 purifiedσC 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 IVσC protein was successfully screened;the prepared mAb could react with ARV GC IVσC 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 1:256000.In conclusion,mAb against ARV GC IVσC protein with good titer and reactivity were successfully prepared in this study,laying a foundation for further researches on the functions of ARV GC IVσC 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 

分 类 号：S855.3[农业科学—临床兽医学]