Apoptosis Of BHK-21 Cells Induced By Rabit Hemorrhagic Disease Virus And Preparation Of McAb Against VP60 Protein

Rabbit hemorrhagic disease virus (RHDV) represents the causative agent of a highly contagious disease in rabbits often associated with liver necrosis, hemorrhages and high mortality. However, the molecular mechanisms involved in RHDV virulence and pathogenicity are not fully understood. It is reported that RHDV could induce the apoptosis of hepatic cells and vascular endothelial cells in vivo, suggesting that the apoptosis of host cells maybe one important pathogenic mechanism of RHDV. The capsid protein VP60 of RHDV has an apparent molecular mass of 60 ku, which has been proved to enhanced neutralizing antibody response in rabbits against RHDV. To develop some effective diagnosis reagents for detecting RHDV and VP60 epitope analysis, we prepared some monoclonal antibodies against RHDV VP60 protein in this research..【Objective】We discussed the apoptosis of BHK-21 cells induced by RHDV in vitro in this research to investigate the characteristic of apoptosis and possible signal pathway. Furthermore, this work can provide theoretical basis for elucidating the pathogenesis of RHDV. Prepare McAbs against VP60 with the recombinant VP60 as antigen and this work settles a foundation for developing some effective diagnosis reagents for detecting RHDV and epitope analysis of VP60.【Method】In this study, the apoptosis of BHK-21 cells infected with RHDV were observed and examined at different time by using DAPI staining, agarose gel electrophoresis and AV-PI staining flow cytometry (FCM). The VP60 was high expressed by the recombinant plasmid pET-30a-VP60 transformed into E.coli, identified and purified, and the recombinant protein was prepared as antigen. Then, BALB/c mice were immunized with the rVP60 for 4 times. According to standard procedure, the hybridomas were prepared by fusing spleen cells from the immunized BALB/c mouse with SP2/0 myeloma cells. Hybridoma cell lines secreting monoclonal antibodies against RHDV were screened by indirect ELISA and subcloning approach. Finaly, the specificity of anti-VP60 McAb was characterized by Western-blotting and indirect immunofluorescence (IFA) respectively.【Results】(1)BHK-21 cells were infected by titration of 300TCID50/mL RHDV. After 24h cultured at 37℃, the cells exhibited apoptosis characteristics of nuclear shrinkage, fragmentation, and appearance of apoptotic bodies; after 48h, 180-200 integer fold sized pieces of DNA Ladders were observed in cells harvested. Besides, it was also demonstrated that the activity of Caspase-3 in infected group was higher than it in control group during the infection process.(2)Two hybridoma cell lines secreting McAbs against RHDV were obtained, named as AE11 and AH4 respectively. It was showed that both of the McAbs could react with the purified RHDV and its rVP60. However, only the McAb AE11 could product the specific fluorescence in RK-13 cells infected with RHDV by IFA.【Conclusion】RHDV can induce apoptosis of BHK-21 cell, and it may be implementation by Up-regulating the Caspase-3 activity. It is possible that the apoptosis in RHD might be important in the development of the pathogenesis of this disease. The McAbs anti-VP60 prepared successfully can react with the rVP60 and purified RHDV antigen.