Liu Y, Zhang Y F, Sun X L, Liu S Y
College of Veterinary Medicine, Inner Mongolia Agricultural University, Huhhot, China.
College of Veterinary Medicine, Inner Mongolia Agricultural University, Huhhot, China
Genet Mol Res. 2016 Aug 29;15(3):gmr8521. doi: 10.4238/gmr.15038521.
The envelope protein (Env) of the Jaagsiekte sheep retrovirus (JSRV) is known to be a unique oncoprotein responsible for inducing ovine pulmonary adenocarcinoma (OPA). The objective of this study was to prepare a specific monoclonal antibody (mAb) against the JSRV Env protein using bioinformatic analysis. According to the structure and epitope prediction results of JSRV Env, the JSRV-Env antigen was prepared via peptide synthesis (amino acid sequence 572-615, denoted as JSRV-Env). BALB/c mice were immunized to prepare the anti-JSRV-Env mAb. Spleen cells were fused with SP2/0 myeloma cells after being screened by indirect ELISA and cloned by limiting dilution. The specificity of mAb was evaluated by western blot analysis and immunohistochemistry assays. Western blot results showed that the JSRV Env protein was able to bind to mAb with high specificity. Immunohistochemistry assays demonstrated that the mAb was able to recognize JSRV Env in adenomatous hyperplasia of the lung. Furthermore, JSRV was detected in peripheral blood leukocytes during the pre-clinical period of OPA in 2 of the 25 sheep using this newly synthesized mAb. Therefore, this mAb may be a useful tool for the detection of JSRV in sheep.
绵羊肺腺瘤逆转录病毒（JSRV）的包膜蛋白（Env）是一种独特的癌蛋白，可诱发绵羊肺腺癌（OPA）。本研究的目的是通过生物信息学分析制备一种针对JSRV Env蛋白的特异性单克隆抗体（mAb）。根据JSRV Env的结构和表位预测结果，通过肽合成制备JSRV-Env抗原（氨基酸序列572-615，记为JSRV-Env）。用其免疫BALB/c小鼠以制备抗JSRV-Env单克隆抗体。经间接ELISA筛选后，脾细胞与SP2/0骨髓瘤细胞融合，并通过有限稀释法进行克隆。通过蛋白质免疫印迹分析和免疫组织化学测定评估单克隆抗体的特异性。蛋白质免疫印迹结果表明，JSRV Env蛋白能够与单克隆抗体高度特异性结合。免疫组织化学测定表明，该单克隆抗体能够识别肺腺瘤样增生中的JSRV Env。此外，使用这种新合成的单克隆抗体在25只绵羊中的2只OPA临床前期外周血白细胞中检测到了JSRV。因此，该单克隆抗体可能是检测绵羊中JSRV的有用工具。
