摘要
为制备抗鸭1型甲肝病毒亚型(DHAV-1a)结构蛋白VP1的单克隆抗体(MAbs),本研究以pGEX-VP1重组蛋白免疫BALB/c小鼠,同时以纯化浓缩的全病毒作为包被抗原,建立了筛选抗VP1蛋白阳性杂交瘤细胞株的间接ELISA方法,经融合、筛选制备杂交瘤细胞及鉴定MAbs的稳定性、特异性、腹水效价和中和活性等生物学活性,获得了2株持续且稳定分泌抗体的杂交瘤细胞(1A2和5G3)。MAbs腹水ELISA效价分别为1∶3.2×104和1∶2.0×106。亚类鉴定均为IgG1/κ型。Western blotting结果显示,2株MAbs均能与DHAV-1aVP1蛋白和DHAV-1a全病毒发生特异性反应。特异性试验结果显示2株MAbs能与鸭1型甲肝病毒(DHAV-1)和DHAV-1a发生交叉反应。中和试验结果显示5G3株具有中和活性。结果表明2株MAbs的ELISA效价高、特异性强、稳定性好,均能与DHAV-1和DHAV-1a全病毒发生特异性反应,其中一株具有中和活性。
To prepare the monoclonal antibodies（MAbs）against DHAV-1a,hybridomas were produced by fusing SP2/0cells with spleen cells from mouse immunized with DHAV-1apGEXVP1 recombinant protein.Two hybridomas stablely secreting MAbs against VP1 protein were identified by indirect ELISA detection with DHAV-1aas coating antigen.The ascetic fluids of MAbs were 1∶3.2×10^4 and 1∶2.0×10^6,respectively.The MAbs were IgG1withκchain.Western blotting analysis showed that the MAbs could recognize the recombinant VP1 protein and DHAV-1a.The neutralization tests showed that one MAb（5G3）had better neutralization activity.Therefore,the results showed that the ELISA titers and specialities of two MAbs were very good,with excellent stability.In addition,they all had cross interaction with DHAV-1and DHAV-1a,one of which had good neutralization activity.
出处
《中国畜牧兽医》
CAS
北大核心
2017年第2期554-560,共7页
China Animal Husbandry & Veterinary Medicine
基金
国家自然科学基金(31472222)
现代农业产业技术体系建设专项(CARS-43)
福建省公益类科研院所专项(2014R1023-3
2015R1023-3和2016R1102)
福建省农业科学院畜牧兽医研究所基金(MYQJ2015-6)
福建省优良蛋鸭品种与设施化养殖创新产业化工程项目(2014-2016)
