Abstract: Mare’k disease (MD) is caused by the infection of Marek’s disease virus (MDV) and is regarded as one of the most serious immunosuppressive and neoplastic diseases of the poultry. Development of specific monoclonal antibodies (mAbs) against MDV is crucial for providing key reagents for the studies on gene functions, pathogenesis and diagnostic techniques. The NE-PER^TM Nuclear and Cytoplasmic Extraction Reagents was used to prepare the nuclear and plasma proteins from the chicken embryo fibroblasts (CEF) cultures infected with vvMDV (very virulent MDV) strain Md5, and then were concentrated by tangential flow ultrafiltration to make the immunogen and immunize female BALB/c mice at age of 6 to 8 weeks. The hybridomas were produced by conventional cell fusion. Through the screening of positive supernatants by indirect immunofluorescence assay (IFA), a total of 31 purified monoclonal hybridomas stably secreting mAbs specific for MDV were obtained, of which 27 hybridomas secret MDV-1 specific mAbs and the other four hybridomas secret conserved mAbs recognizing MDV-1, MDV-2 and herpesvirus of turkey (HVT). Furthermore, the J-1F3-C6 was identified as a specific mAb against MDV glycoprotein B (gB) thorough the IFA staining of 293T cells over-expressing gB protein. The titer of mAb J-1F3-C6 in ascites is 1∶25 600 determined by IFA, and the light chain and IgG subtype was characterized as Kappa and IgG2a, respectively. The cross reaction experiments have shown that the J-1F3-C6 specifically recognized the gB proteins from all the tested strains of MDV-1, MDV-2 and HVT, but for Western blot analysis it can only recognize MDV-1 and HVT strains. In conclusion, we have developed a pool of mAbs against MDV and identified a mAb specific for gB protein, providing a solid foundation for future research.

Key words: Marek’s disease, monoclonal antibody, glycoprotein gB, IFA, confocal analysis, Western blot