Preparation Of Monoclonal Antibody Against GB Protein Of Pseudorabies Virus Named HeN1 And Identification Of The B-cell Epitope

Pseudorabies is caused by pseudorabies virus that can cause symptoms of fever,itch and encephalomyelitis in many kinds of livestock and wild animals.Since 1947,the disease has been imported into China,which has been controled by using the vaccines.However,since 2011,new epidemic strains emerged in many vaccinated pig farms in China,which indicates the vaccine does not provide complete protection against these strains.Therefore,it is necessary to monitor pseudorabies and explore its structure.gB glycoprotein is the most conservative protein of the porcine pseudorabies virus,and the antibodies against it could represent the antibody level of the swinery.The aims of this experiment were to prepare monoclonal antibodies against PRV gB protein and identify its antigenic epitope.Six-week old BALB/c mice were immunized with ultracentrifugation-purified PRV HeN1 strain.The myeloma cells SP2/0 fused with spleen cells of the positive mouse.Positive hybridoma cells were screened using indirect immunofluorescence assay?IFA?,and a hybridoma cell line 1E7 against PRV HeN1 gB was obtained by four-times subcloning.Western blot showed that 1E7 MAb specifically reacted with both PRV and gB protein expressed by eukaryotic.The MAb belongs to IgG2b subtype with?chain.Blocking ELISA showed that 1E7 could be blocked by the anti-serum of PRV.In addition,the titers of the MAb in cell culture medium and ascites were 1:160 and 1:12800,respectively.Finally,a series of overlapping sequeces covering gB proteins were expressed by prokaryotic expression system and the sequence of the epitope MAb was determined as S⁸¹AEESLE⁸⁷,which were relatively conservative among PRV strains.The MAb prepared in this study was propitious to develop detected method against antibody and research the structure and function of gB protein further.