杨少华　张琳　崔宁　黄庆华　黄艳艳　许传田

摘要：本试验以纯化的新城疫病毒（NDV）弱毒株D58为免疫原免疫6～8周龄BALB/c小鼠，分离脾脏淋巴细胞与小鼠骨髓瘤细胞进行融合，经多次ELISA鉴定和血凝抑制试验（HI）筛选和亚克隆培养，获得了4株稳定分泌抗体的杂交瘤细胞并制备了单抗。经鉴定，4株单抗均有ELISA效价，其中1株单抗有中和活性和HI活性，只与ClassⅠNDV反应，与ClassⅡNDV不反应。该单抗与不同群NDV反应的差异性为NDV的鉴别诊断和不同毒株抗原差异性研究奠定了一定基础。

关键词：鸡;新城疫病毒;单克隆抗体;HN蛋白

中图分类号：S852.65+7文献标识号：A文章编号：1001-4942（2019）10-0135-04

Preparation of Monoclonal Antibody against

Class Ⅰ Newcastle Disease Virus

Yang Shaohua， Zhang Lin， Cui Ning， Huang Qinghua， Huang Yanyan， Xu Chuantian

（Institute of Animal Science and Veterinary Medicine， Shandong Academy of Agricultural

Sciences/Shandong Key Lab of Animal Disease Control and Breeding， Jinan 250100， China）

Abstract The lentogenic NDV D58 strain purified by differential centrifugation was immunized to 6～8-week BALB/c mice. The spleen cells of the mice were hybridized with SP2/0 three days after the last immunization. After screened by indirect ELISA and HI assay， and cloned by limiting dilution， four hybridoma cells secreting antibodies stably were obtained and the monoclonal antibodies were prepared. All four McAbs had ELISA titers， and one of them had neutralization and HI activities and could react with ClassⅠNDV specifically. The differences of the reaction between McAb and different groups of NDV lays a foundation for the clinical identification of NDV and the differential study of antigens in different strains of NDV.

KeywordsChicken; Newcastle disease virus; Monoclonal antibody; HN protein

新城疫（Newcastle disease，ND）是由新城疫病毒（Newcastle disease virus，NDV）引起的一種禽的急性、高度传染性疾病，国际兽疫局（OIE）将其列为动物A类疫病[1，2]。自1926年被确诊以来，新城疫在世界范围内广泛传播，目前我国由于疫苗的广泛使用，新城疫的发病率逐年降低，但非典型性ND仍零星散发，ClassⅠ弱毒株在禽群中广泛存在[3-5]。ClassⅠ毒株在鸡群中存在毒力返强和散毒的风险，因此有必要加强鸡群中此类毒株的监测。

尽管NDV只有一个血清型，但不同亚型的病毒间存在较大的抗原差异。单克隆抗体具有针对单个抗原决定簇的特异性，可以识别不同的抗原位点，因此可以用来检测不同毒株之间的抗原差异。本研究以纯化的ClassⅠNDV弱毒株为免疫原，拟研制具有中和活性和血凝抑制活性的单抗，以期为NDV的鉴别诊断和抗原差异研究提供参考。

1 材料与方法

1.1 试验材料

新城疫ClassⅠ弱毒株D58（KF055276）及其它NDV分离株由本实验室分……