Sun Miao, Yu Duo, Mo Hongfei, Cao Hong, Chen Chen, Chen Fuyong
College of Veterinary Medicine, China Agricultural University, Beijing 100193, China.
J Vet Med Sci. 2012 Jun;74(6):693-7. doi: 10.1292/jvms.11-0428. Epub 2011 Dec 28.
Avian leukosis virus subgroup J poses a great threat to the poultry industry in China. To reduce the economic losses, a quick method for detection of ALV-J antigen is required for diagnosis and identification of the congenitally transmitting hens. In this study, we report the production and evaluation of one monoclonal antibody (MAb) suitable for achieving these goals. The gp85 gene of avian leukosis virus subgroup J CAUHM01 China isolates was subcloned into the expression vectors pGEX-6p-1 and pET28a and successfully expressed in E. coli. After immunizing BALB/c mice with recombinant His-Jgp85 protein, splenic cells from immunized mice were fused with SP2/0 myeloma cells to produce hybridomas. We isolated and characterized one ALV-J gp85-specific MAb by determining its titer, affinity and IgG subclass. In addition, we performed epitope mapping and determined the epitope for the MAb 1E3 to be 81-92 aa of ALV-J gp85 protein (LPWDPQELDILG). Bioinformatics analysis and IFA studies revealed that this epitope is conserved among all ALV-J isolates and that this antibody could serve as a useful reagent for ALV-J detection and diagnosis.
J亚群禽白血病病毒对中国家禽业构成了巨大威胁。为减少经济损失，需要一种快速检测ALV-J抗原的方法来诊断和鉴定先天性传播母鸡。在本研究中，我们报告了一种适合实现这些目标的单克隆抗体（MAb）的制备和评估。将J亚群禽白血病病毒中国CAUHM01分离株的gp85基因亚克隆到表达载体pGEX-6p-1和pET28a中，并在大肠杆菌中成功表达。用重组His-Jgp85蛋白免疫BALB/c小鼠后，将免疫小鼠的脾细胞与SP2/0骨髓瘤细胞融合以产生杂交瘤。我们通过测定其效价、亲和力和IgG亚类，分离并鉴定了一种ALV-J gp85特异性单克隆抗体。此外，我们进行了表位作图，确定单克隆抗体1E3的表位为ALV-J gp85蛋白的81-92位氨基酸（LPWDPQELDILG）。生物信息学分析和免疫荧光分析研究表明，该表位在所有ALV-J分离株中保守，并且该抗体可作为ALV-J检测和诊断的有用试剂。
