新型鸭呼肠孤病毒σB蛋白单克隆抗体的制备及其抗原表位的鉴定  

Preparation of the monoclonal antibodies againstσB protein of novel duck reovirus and identification of the antigenic epitope

作　　者：华炯钢[1] 叶伟成[1] 倪征[1] 陈柳[1] 朱寅初 云涛[1] 张存[1] HUA Jiong-gang;YE Wei-cheng;NI Zheng;CHEN Liu;ZHU Yin-chu;YUN Tao;ZHANG Cun(Institute of Animal Husbandry and Veterinary Sciences,Zhejiang Academy of Agriculture Sciences,Hangzhou 310021,China)

机构地区：[1]浙江省农业科学院畜牧兽医研究所,浙江杭州310021

出　　处：《中国预防兽医学报》2022年第4期428-434,共7页Chinese Journal of Preventive Veterinary Medicine

基　　金：浙江省重点研发计划项目(2015C02012、2019C02052);浙江省农业重大技术协同推广项目(2021XTTGXM04-02);浙江省农业科学院专项经费项目(2022R22CB003)。

摘　　要：B蛋白是新型鸭呼肠孤病毒(NDRV)的外衣壳蛋白之一,可诱导宿主机体产生群特异性抗体。为制备NDRVσB蛋白的单克隆抗体(MAb),并鉴定其抗原表位,本研究利用原核表达后纯化的重组σB蛋白(rσB)免疫BALB/c小鼠,取其脾淋巴细胞与SP2/0细胞融合,以纯化的rσB为包被抗原,通过间接ELISA方法筛选出3株杂交瘤细胞,其分泌的MAb分别命名为10-A5、A1-A9和B7-E5。将NDRV ZJ00M株以MOI 0.01感染DF-1细胞72 h后收获细胞,通过western blot鉴定获得的MAb与天然表达的σB蛋白的反应性;分别将NDRV ZJ00M株、鸭经典呼肠孤病毒(CDRV)ZJ2000M株和鸡呼肠孤病毒(ARV)S1133株均以MOI 0.01感染DF-1细胞,48 h后经间接免疫荧光试验(IFA)鉴定MAb的交叉反应性。Western blot结果显示,3株MAb均在41 ku出现特异性条带,表明其均能够与NDRVσB蛋白发生特异性反应;IFA结果显示:3株MAb均与NDRV反应,而不与CDRV和ARV发生交叉反应。上述结果表明,本研究制备的MAb反应原性和特异性均较强。利用人工合成的37条NDRVσB蛋白多肽及其截短的多肽,采用肽扫描法结合间接ELISA方法对3株MAb识别的抗原表位进行鉴定,并通过抗原表位氨基酸序列比对,分析该抗原表位在禽正呼肠孤病毒各成员中的保守性。结果显示,3株MAb识别的最小抗原表位均为33DIEEFHTPDVI43,且该抗原表位氨基酸序列在不同地域和不同宿主来源的NDRV分离株间的保守性均较高。本研究首次制备了NDRVσB蛋白的MAb,并鉴定了其抗原表位及该表位的保守性,为NDRV检测方法和表位标记疫苗的研究奠定基础。B is one of the major outer capsid proteins of novel duck reovirus(NDRV),which is able to induce group-specific antibodies against the virus.In order to prepare monoclonal antibody(MAb)against NDRVσB protein and identify its epitope,BALB/c mice were immunized with purified recombinantσB protein(rσB).Spleen lymphocytes were fused with SP2/0 cells.Using purified rσB as coating antigen,three hybridoma cell lines were screened by indirect ELISA.The secreted MAb was named 10-A5,A1-A9 and B7-E5,respectively.DF-1 cells were infected with NDRVZJ00M strain at a dose of MOI 0.01,and the cells were harvested 72 hours later,and the reactivity of MAb with naturally expressedσB protein was detected by western blot.DF-1cells were infected with NDRVZJ00M strain,duck classical reovirus(CDRV)ZJ2000M strain and chicken reovirus(ARV)S1133strain at the dose of MOI 0.01,respectively,and the cross-reactivity of MAb was identified by indirect immunofluorescence assay(IFA)48 hours later.The results of western blot showed that the three strains of MAb could react specifically with theσB protein of the whole NDRV virus and showed a specific band in 41ku,while the results of IFA showed that all the three strains of MAb reacted with NDRV,but not with classical duck reovirus(CDRV)and avian reovirus(ARV).The above results indicated that the MAb obtained in this study had strong reactivity and specificity to NDRV.Using 37 synthetic peptides of NDRVσB protein and their truncated peptides,the antigenic epitopes recognized by three MAbs were identified by peptide scanning and indirect ELISA,and the conservatism of the antigenic epitopes in the members of avian orthoreovirus was analyzed by sequence alignment.The results showed that the smallest antigenic epitope recognized by the three MAbs was33DIEEFHTPDVI43,and the antigenic epitope was highly conserved among NDRV isolates from different regions and different host sources.In this study,MAb of NDRVσB protein was prepared for the first time,and its antigenic epitope and its conservation we

关 键 词：新型鸭呼肠孤病毒 σB蛋白 单克隆抗体 抗原表位 

分 类 号：S852.65[农业科学—基础兽医学]