交叉中和血清1型和血清3型鸭甲肝病毒单克隆抗体的制备与鉴定

鸭甲型肝炎（duck hepatitis A，DHA）是危害养鸭业的主要疾病之一，是一种雏鸭的急性、高度致死性传染病。本研究旨在研制一种对我国流行的鸭甲型肝炎血清1型（DHAV-1）和血清3型（DHAV-3）病毒具有交叉中和作用的单克隆抗体（monoclonal antibody，McAb）。人工合成12条DHAV-1和DHAV-3的VP1蛋白共有的抗原表位肽，分别与钥孔血蓝蛋白（keyhole limpet hemocyanin，KLH）载体偶联作为免疫抗原，免疫BALB/c小鼠制备McAb。通过检测McAb与DHAV-1和DHAV-3的交叉反应性，测定McAb对病毒增殖的抑制效率、中和效价以及攻毒保护率等来筛选McAb。本研究共获得了12株稳定分泌McAb的杂交瘤细胞株，其中6株（C1、C4、C7、C12、C13、C16）分泌的McAb同时与DHAV-1和DHAV-3发生特异性交叉反应； C1、C4、C7、C16可以抑制DHAV-1和DHAV-3在鸭胚中的增殖，抑制率在75.34%～100.00%不等；对DHAV-1和DHAV-3的中和效价：C1（1：3&1：5）、C4（1：3&1：3）、C7（1：10&1：11）和C16（1：9&1：9）；C7和C16对DHAV-1和DHAV-3攻毒雏鸭的保护率较高，分别为“70%、80%”和“100%、60%”。本研究成功研制出对DHAV-1和DHAV-3具有交叉中和活性的McAb 4株，其中2株对病毒的感染具有良好的预防效果，为DHA的防控提供了新材料和新思路。

Preparation and Identification of Monoclonal Antibodies with Cross-neutralizing Activity against Duck Hepatitis A Virus Serotype 1 and 3

Duck hepatitis A (DHA), an acute and highly lethal infection in ducklings, is one of the main diseases threatening the duck industry. The purpose of this study was to develop a monoclonal antibody (McAb) that has cross-neutralization activity against two most prevalent serotypes of DHAV in China, DHAV-1 and DHAV-3. Twelve epitope peptides shared by VP1 proteins of DHAV-1 and DHAV-3 were synthesized artificially and respectively conjugated with keyhole limpet hemocyanin (KLH) protein. BALB/c mice were then immunized with those antigens and McAbs were prepared. The obtained McAbs were screened by detecting the cross-reactivity with DHAV-1 and DHAV-3, the inhibitory efficiency on virus proliferation, the neutralization titer, and the protection rate against virus infection. A total of 12 hybridomas were obtained, the McAbs secreted by 6 hybridomas (C1, C4, C7, C12, C13, C16) in those had specific cross-reactions with DHAV-1 and DHAV-3. The McAbs secreted by hybridomas (C1, C4, C7 and C16) had higher inhibitory efficiency on DHAV-1 and DHAV-3 proliferation in duck embryos, ranging from 75.34% to 100.00%. The neutralization titer of McAbs secreted by hybridomas (C1, C4, C7 and C16) against DHAV-1 and DHAV-3 were 1:3 & 1:5, 1:3 & 1:3, 1:10 & 1:11, 1:9 & 1:9, respectively. The protection rate of McAbs secreted by hybridomas (C7 and C16) against DHAV-1 and DHAV-3 infection were 70% & 80%, 100% & 60%, respectively. In this study, four McAbs with cross-neutralizing activity against DHAV-1 and DHAV-3 were developed, two of those had a higher protection rate against virus infection, providing new materials and ideas for the prevention and control of DHA.