Preparation Of Monoclonal Antibody Against Pseudorabies Virus UL31 Protein

Pseudorabies(PR)is an acute swine infection caused by Pseudorabies virus(PRV).PRV can infect pigs of all ages.The main manifestations of PRV in pigs are fever,anorexia,respiratory symptoms,itching of skin,reproductive dysfunction of pregnant sows,and antinomy of piglets.Moreover,PRV is infected by a wide range of hosts,such as cattle,sheep,wild boar,fox,raccoon,dog,rabbit,rodent,etc.,and human cases of PRV have been reported,and PRV has the risk of cross-species transmission and infection to humans.China has listed pseudorabies as the second-class animal blight,so the pathogenic mechanism of PRV needs further study.In the study of pseudorabies virus,it was found that UL31 protein interacts with UL34 protein to form a complex located in the nuclear membrane,which plays an important role in the nucleation of PRV after completion of replication.However,the mechanism of action of U31 protein has not been fully clarified so far.Therefore,the study of monoclonal antibody against UL31 protein was carried out to lay the foundation for the study of UL31 protein.In order to prepare monoclonal antibody to PRV UL31 protein,this study first used the prokaryotic expression system to prepare HIS-UL31 recombinant protein,and the purified HIS-UL31 recombinant protein was identified by Western blot.The recombinant protein of HIS-UL31 could bind specifically to His monoclonal antibody.His-UL31 protein was used as the coated antigen and the coated concentration was 8μg/m L.The serum dilution of PRV-infected pig was 1:25600.An indirect ELISA method was developed to detect UL31 antibody.His-UL31 was used as antigen to immunize 12-week-old female BALB/c mice.After three times of immunization,spleen cells were extracted from the mice and fused with SP2/0 cells.After four indirect ELISA screening,a hybridoma cell strain with stable secretion of monoclonal antibody against UL31 protein was obtained and named UL31 5-11.The number of chromosomes in SP2/0 cells and UL31 5-11 monoclonal cells was counted,and the average number of chromosomes in UL31 5-11 monoclonal cells was about 65,which was the sum of the number of chromosomes of the two parents.The subtype of monoclonal antibody was identified by the supernatant of hybridoma cells and identified as Ig M.5×106 monoclonal cells per mouse were injected intraperitoneally to obtain a large amount of ascites with UL31 5-11 antibody.Ascites were purified by caprylic acid and low temperature ethanol precipitation method to obtain MAb 5-11.The titer of monoclonal antibody was determined by indirect ELISA method to be1:5120.Indirect immunofluorescence assay,western blot analysis and immunohistochemical assay confirmed that MAb 5-11 had good specificity and reactivity.In this study,monoclonal antibody to PRV UL31 was successfully prepared,which laid a foundation for further study on the function and structure of UL31 protein.