Preparation Of Monoclonal Antibodies Against Glycoprotein B And Immunogenicity Of Infectious Bovine Rhinotracheitis Virus

Infectious bovine rhinotracheitis(IBR)is caused by Infectious bovine rhinotracheitis virus(IBRV),which is characterized by reduced production of milk yield,fertility and working in cattle,and causes great economic losses to the global cattle industry.The glycoprotein B(g B)of IBRV,a highly conserved glycoprotein within the herpesvirus family,is located in the viral capsid,and plays an essential role in the mechanisms of virus transmission,spread and fusion.In this study,the characteristics of the g B protein,such as signal peptide cleavage sites,antigen epitopes,transmembrane regions and hydrophobicity were analyzed by bioinformatics.The target gene sequence does not exist in the transmembrane region and signal peptide cleavage site of the g B gene,which is characterized by strong hydrophilicity and positive separation of antigenic determinants.With the use of IBRV NM14 of laboratory isolated as template,the prokaryotic recombinant expression plasmids p ET-32a-g B and eukaryotic expression plasmids pc DNA3.1-g B for truncated g B gene was successfully constructed,and the recombinant plasmids were able to express the g B proteins in E.coli.Meanwhile,preparation of monoclonal antibody against recombinant g B protein and subunit vaccine of IBRV.Furthermore,subunit vaccine potency testing against IBRV g B was studied in guinea pig models.The result showed that prokaryotic expression system was employed to produce a large quantity of recombinant g B inclusion bodies have good immunogenicity.We successfully generated four mice anti-g B monoclonal antibodies has a good spatial configuration,which could specifically bind with recombinant plasmids expression of g B proteins,and virus antigen of IBRV.The expression levels of neutralizing antibody and specific g B antibody was detected by neutralization test and indirect ELISA.The subunit vaccine was immunized to guinea pigs,we found that the expression levels of neutralizing antibody and g B antibody significantly higher than those in the blank control group(P < 0.001),and at 35 days immunization the antibody level reached the peak,which the neutralizing titer could reach 1:85.After attacking the IBR wild virus strain,immunization with a recombinant subunit vaccine group showed without obvious clinical symptoms,and their clinical score lower than the unimmunized with a recombinant subunit vaccine group.In addition,compared with immunization subunit vaccine groups,the unimmunized subunit vaccine groups the high level of virus in the lung tissue of guinea pigs(P < 0.001).The lung tissue of the guinea pig models developed typical interstitial pneumonia lesions upon infection with the IBR wild strain,however,the lung tissues of guinea pigs immunized with the subunit vaccine developed only milder lesions after the wild virus challenge,these findings align with those observed in the results of tissue viral load.Altogether,the g B subunit vaccine could effectively block virus replication and protect cohabiting guinea pigs from infection.This study provides a foundation for the application of g B protein in the clinical diagnosis of IBRV or in the vaccination against IBRV in the future.