Preparation Of Monoclonal Antibodies Against Infectious Bovine Rhinotracheitis Virus And Identification Of Antigen Epitopes With Partial Monoclonal Antibodies

Infectious bovine rhinotracheitis virus,belonging to the?herpes virus subfamily,also called Bovine herpesvirus 1?BHV-1?,is one of the major pathogen from cattle,can cause some diseases of cattle such as infectious bovine rhinotracheitis?IBR?,miscarriage,conjunctivitis,infections in newborn calves,abortion,infectious pustular vulvovaginitis?IPV?and immunosuppression.Infectious bovine rhinotracheitis?IBR?is an acute and contagious infectious disease and distributed worldwide.The disease has a low mortality rate,but affects the growth of animals and has serious restrictions on milk production and international trade,resulting in significant economic losses.The unique feature of the disease is that BHV-1 establishes latent infection in the sensory neurons such as the trigeminal ganglion after acute infection.The latent virus can be periodically activated and widely spread in the herd,making the disease difficult to eradicate.If cattle would be immunized with gE-deleted vaccine of BHV-1,we could use the monoclonal antibodies against gE protein of BHV-1 to establish the corresponding gE-ELISA,which could be used to distinguish vaccinated and infected cattle to achieve the purpose of eradicating IBR.Therefore,the preparation of monoclonal antibodies against gE protein and the main glycoprotein of BHV-1 is of great significance to the diagnosis and prevention of IBR.Two methods were used to immunize BALB/c mice.One method was to immunize mice with whole virus,and another method was to immunize mice with gE-deleted BHV-1 while with cyclophosphamide?CP?and subsequent immunization with whole BHV-1.The hybridoma cells were screened by indirect ELISA with purified BHV-1 and gE-deleted virus coated ELISA plates.By screening,ten monoclonal antibodies against BHV-1 were prepared.Indirect immunofluorescence results showed that these MAbs reacted with BHV-1 and gE-deleted BHV-1.The results of the identification of monoclonal antibody subclasses showed that the subtypes of six MAbs were IgM/?,three were IgA/?and one was IgG2b/?.Western blot analysis showed that ten MAbs had no reaction band with BHV-1 in Western blotting,indicating that the antigen epitopes of the ten monoclonal antibodies were conformational.At the same time,two hybridoma cell lines 3C2 and 1F5 secreting monoclonal antibody against VP8 protein and one hybridoma cell line 5B7 secreting monoclonal antibody against gD protein of BHV-1 were used for the identification of antigen epitopes.Firstly,BALB/c mice were inoculated with the hybridoma cell lines to prepare ascites,and then the antigen epitopes were determined by peptide scanning technique with monoclonal antibodies.The results showed that the antigen epitope of MAb 3C2 and MAb 1F5 against VP8 protein were ¹³⁸PHRSLLERTA¹⁴⁷ and ¹⁸³GGGQEPG¹⁸⁹,respectively.The antigen epitope of MAb 5B7 against gD protein was ³³⁶PEGWPSL³⁴².The antigen epitopes of MAb 3C2,1F5 and 5B7 were linear.Further analysis showed that the antigen epitopes of the three strains of MAbs were highly conserved among different BHV-1 isolates.In summary,ten MAbs against BHV-1 were prepared and their antigen epitopes were all conformal.By peptide scanning technique,the antigen epitopes of three MAbs established in our laboratory were determined to be linear and highly conserved in different BHV-1 isolates.This study laid a foundation for the diagnosis of BHV-1,the study of the structure and function of VP8 protein and gD protein and the subsequent preparation of gE monoclonal antibodies against BHV-1.