The Development Of McAbs Against Pseudorabies Virus P UL37,VP16,VP22 And Establishment Of Immunogold Labeling Methods

Pseudorabies,caused by pseudorabies virus(PRV),is an acute infectious disease in various mammals.Sows are the natural hosts of the virus.PRV infections may result in death of the fetuses,abortion in sows and acute death in piglets.PRV belongs to the family Herpesviridae,and has the typical four lays structure.The tegument,lying between the capsid and the envelope,composes a layer of redundant proteins.PUL37,VP16 and VP22 encoded by UL37,UL48,UL49 separately,play diverse roles in the viral replication and assembly.Study on these proteins is of great significance to exploring new antiviral mechanism for against the PRV infections.However,methods for the detection and localization of those three proteins in PRV infected cells have not been established yet.To develop monoclonal antibodies(McAbs),the UL49,UL48,UL37 genes were subcloned separately into pCAGGS and the expressed prokaryotic proteins were purified by gel slices after KCl staining.The prepared plasmids and purified proteins were used as immune materials and injected intramuscularly into 6-week-old Balb/c female mice.Three days after the last immunization,the cell fusion experiments were performed by fusing SP2/0 cells with spleen cells of BALB/c mice immunized.Six strains of McAbs were obtained and named 3D6(VP22),1D4(VP16),4B6(VP16),1E2(pUL37),2G2(pUL37),and 11C3(pUL37),respectively.Both the assays,indirect immunefluorescence(IFA)and Western-blot,showed that the selected McAbs could specifically react with the corresponding proteins.The subclass of all those McAbs were proved to belong to IgM?.The titers of the McAbs 1E2,2G2,1D4,11C3,4B6,and 3D6 were determined to be 1:32000,1:32000,1:8000,1:6000,1:4000,1:4000,and 1:3000,respectively.PRV strain HLJ 2013 was inoculated into susceptible cells PK15 at 0.1 MOI.Sixteen hours after the inoculation,the infected cells were collected and ultrathin section samples were prepared for the following immunogold labeling experiments.The McAbs obtained from above experiments were at dilutions of 1:50,1:100,and 1:200 as primary antibodies,and commercialized standard anti-mouse gold labeled IgG(1:100 dilution)were used as secondary antibodies.The VP22,VP16 and pUL37 proteins expressed in infected PK15 cells were detected by electron microscopy(EM).Analyses of the EM images showed that McAbs 3D6,1D4 and 1E2 showed high specificity for labeling VP22,VP16,and pUL37,respectively,and the optimized dilutions for immunogold labeling methods were 1:100(3D6 / 1D4),and 1:200(1E2).In summary,McAbs against pUL37,VP16 and VP22 were developed in this study.Using these McAbs as primary antibodies,we also established and optimized the immunogold labeling methods for detecting the three proteins VP22,pUL37,VP16 in PRV infected cells,which provides technical support for studying functions of PRV VP22,pUL37,VP16 and the mechanism of PRV tegument assembly.