Zha Mei, Yang Jing, Zhou Linlin, Wang Hongren, Pan Xing, Deng Zhaomin, Yang Yuan, Li Wanyi, Wang Baoning, Li Mingyuan
Department of Microbiology, West China School of Basic Medical Sciences and Forensic Medicine, Sichuan University, Chengdu, Sichuan 610041, P.R. China.
Institute of Liver Diseases, Renmin Hospital, Hubei University of Medicine, Shiyan, Hubei 442000, P.R. China.
Exp Ther Med. 2019 Aug;18(2):1384-1390. doi: 10.3892/etm.2019.7708. Epub 2019 Jun 25.
The aim of the current study was to prepare and identify mouse anti-human rotavirus (RV) VP7 monoclonal antibodies and explore their protective effects on RV infection. The mouse anti-human RV VP7 monoclonal antibody was produced using the ascites method and identified via western blot analysis. neutralization of mouse anti-human RV VP7 monoclonal antibodies was detected by performing an MTT assay. The TCID value was calculated to obtain antibody neutralization titers. A mouse RV infection model was generated to assess the protective effect of the mouse anti-human RV VP7 monoclonal antibody in experimental animals. Monoclonal antibodies were successfully prepared and their purity reached ≥90%. Western blotting demonstrated that monoclonal antibodies specifically bound to the purified Wa RV strain, with a specific reaction band at ~40 kDa. Monoclonal antibody neutralization results demonstrated that cell survival rate in the virus + monoclonal antibody group was higher than that in virus + maintenance fluid group (P<0.05). Monoclonal antibody neutralization titer detection revealed that the cytopathic effect did not extend beyond 4 days. In addition, the calculated monoclonal antibody neutralization titer was 1:446. The results revealed that the positive rate of colloidal gold RV in the 100 µl monoclonal antibody group was significantly lower than that in the control group (P<0.05). Furthermore, the protection rate of the 100 µl monoclonal antibody group was 71.4%, whereas the 50 µl monoclonal antibody group was 42.9% and the ribavirin group was 57.1%. In conclusion, the results of the current study demonstrated that mouse anti-human RV VP7 monoclonal antibodies can be successfully prepared using ascites method. These antibodies also effectively neutralize the cytotoxic effects of the human RV Wa strain and mouse anti-human RV VP7 monoclonal antibodies also exhibited a good protective role in mice. Furthermore, greater protective effects were observed at a higher dose and the protective effects of these high dose treatments were superior to that of ribavirin.
本研究的目的是制备并鉴定小鼠抗人轮状病毒（RV）VP7单克隆抗体，并探讨其对RV感染的保护作用。采用腹水法制备小鼠抗人RV VP7单克隆抗体，并通过蛋白质印迹分析进行鉴定。通过MTT法检测小鼠抗人RV VP7单克隆抗体的中和作用。计算半数组织培养感染剂量（TCID）值以获得抗体中和效价。建立小鼠RV感染模型，以评估小鼠抗人RV VP7单克隆抗体在实验动物中的保护作用。成功制备了单克隆抗体，其纯度达到≥90%。蛋白质印迹显示单克隆抗体与纯化的Wa RV株特异性结合，在约40 kDa处有特异性反应条带。单克隆抗体中和结果表明，病毒+单克隆抗体组的细胞存活率高于病毒+维持液组（P<0.05）。单克隆抗体中和效价检测显示细胞病变效应在4天内未扩展。此外，计算得到的单克隆抗体中和效价为1:446。结果显示，100 μl单克隆抗体组胶体金RV阳性率显著低于对照组（P<0.05）。此外，100 μl单克隆抗体组的保护率为71.4%，而50 μl单克隆抗体组为42.9%，利巴韦林组为57.1%。总之，本研究结果表明，采用腹水法可成功制备小鼠抗人RV VP7单克隆抗体。这些抗体还能有效中和人RV Wa株的细胞毒性作用，并且小鼠抗人RV VP7单克隆抗体在小鼠中也表现出良好的保护作用。此外，在较高剂量下观察到更强的保护作用，且这些高剂量治疗的保护作用优于利巴韦林。
