Development Of Monoclonal Antibodies Against Marek's Disease Virus And The Expression And Purification Of MEQ Protein

Marek's disease(Marek's,Disease,MD)by Marek's disease virus(Marek's Disease Virus,MDV)infection of chickens caused by a highly contagious,infectious and lymphoproliferative tumors,MD can cause immune suppression and other disease susceptibility,causing huge economic losses to the world poultry industry.MDV is mainly in the diameter of 85~100 nm naked virus particles exist in cells,with strict a cells combined with herpes simplex virus,virus particles with intact membrane,size is about 273~400 nm,does not have the cell binding,which can independently of cell survival,this is the main reason of the disease continued spread and outbreak.In this study,the MDV superstrong strain GX0101 was purified from chicken embryo fibroblast(CEF)as immunogen,and the monoclonal antibody specific to identify MDV was prepared.GX0101 CEF infection,96 h cells were collected after repeated freezing and thawing 3 times,the selection of 100 k D tangential flow ultrafiltration centrifugal tube to be concentrated on peak virus concentrate to 5 m L,after centrifugation the supernatant by Sepharose 4 Fast Flow(4FF)as gel chromatography medium separation and purification of virus particles,polymerase chain reaction(PCR)and real time fluorescent quantitative PCR(q RT-PCR)analysis of different collection peak MDV levels,and through transmission electron microscopy(TEM)observed MDV naked virus particle diameter of about 100 nm.The selection of 100 k D tangential flow ultrafiltration centrifugal tube concentrated to obtain peak virus concentrate by immune Balb /c female mouse monoclonal antibody.Through immunoperoxidase monolayer cell test(IPMA)and Western blot screening,107 positive clones were finally obtained.Among them,1 strong positive Mc Ab hybridoma cells 10B2 prepared by monoclonal antibody ascites IPMA titer was 1:2.56×106.The results of this study have laid an important foundation for the screening,identification,preparation and the research and development of the diagnostic reagents for the subsequent MDV Mc Abs.MEQ protein as the mostworthy Oncogenic protein is very important for the study of oncogenousmechanism in MDV.MEQ protein was expressed by p ET-30 a vectorin Rosetta cell.The results reveal that protein was in supernatant.MEQ was purified by Ni column affinity chromatography.MEQ laid the foundation for preparation of monoclonal antibodies.