Evaluation Of B-cell Epitopes Of GB Protein And Development Of Blocking ELISA With Monoclonal Antibody For Detection Of Antibody To PRV

Pseudorabies(PR),caused by pseudorabies virus(PRV),is an important infectious disease in pigs,cattles,sheeps,and other mammals.The pig is the only host that can survive after PRV infection,namely PRV storage hosts.Pseudorabies bringing huge economic losses to the global pig industry has attracted global attention.The main clinical symptoms include:central nervous system disorders in newborn piglets,respiratory symptoms in piglets and fattening pigs,reproductive disorders in pregnancy.Currently,the anti-viral vaccination has become the primary means of prevention and control of pseudorabies.However,since 2011,a severe PRV outbreak have taken place in many PRV vaccinated pig farms,and caused great economic losses.The effective and rapid method for the detection of PRV is far-reaching for the control and eradication of PRV.In this study,we isolated a virulent strain of PRV from an immunized pig farm and made 4 monoclonal antibodies against gB and gE proteins.In addition,a linear epitope was identified in gB protein.And a blocking ELISA method for PRV was developed based on the recombinant gB protein and its specific monoclonal antibodies.The main contents of the researchs were as following:1.Isolation and pathogenicity of a highly virulent pseudorabies virus in Jangsu provienceA strain named PRV NJ was isolated from clinical infected pigs.The sequence analysis of the gC and gD genes showed that this isolate contained 7 and 2 amino acid insertions respectively.The results of phylogenetic tree ananlysis indicated that it belonged to a relatively independent cluster and has a close phylogenetic relationship with the isolates from Asia.The 100-day-old piglets free of PRV infection were challenged with PRV NJ strains and LA strains,respectively.The results showed that pigs inoculated intranasally with PRV NJ strains were characterized by 100%mortality within 7 days after challenge,significant clinical symptoms and histopathological changes,while pigs inoculated intranasally with LA strains were characterized by only mild respiratory symptoms,temperature changes and slight histopathological changes.It indicated that the virulent of PRV NJ strain enhanced significantly.2.Preparation and identification of monoclonal antibodies against gB and gE proteins of PRVIn this study,the gB and gE recombinant protein of the strong virulent strain ZJ01 were obtained by the E.coli expression system,which were used to immune BALB/c mices.Four monoclonal antibodies(McAbs)were prepared by cell fusion,cloning and indirect enzyme-linked immunosorbent assay(ELISA).The ELISA titers in supernatant were 1:1600?1:6400,and titers in asicite were more than 1:400000.The isotypes of B1B6 and B3D7 MAbs against gB protein belong to IgG2b subtype with ? chain,and E1B11 and E5C10 MAbs against gE protein belong to IgG1 subtype with ? chain.Western blot and IFA results showed that the four MAbs could react specifically with PRV wild strains ZJ01,HZ,NJ,SD and LA.While B1B6 and B3D7 hybridoma cell lines not only reacted with PRV wild strains but also reacted with PRVgE-strain Bartha-K61.However,E1B11 and E5C10 could not react with PRVgE-strain Bartha-K61.Those monoclonal antibodies provide valuable tools for further development of antigen epitope analysis and specific diagnosis techniques.3.Identification of B cell epitop of monoclonal antibodies against gB proteins of PRV ZJ01Seven truncated gB protein genes were expressed successfully as His-fusion protein in E.coli.system to determine epitops of monoclonal antibodies(McAbs)against gB protein of PRV ZJO1.Western blot and epitope mapping results indicated that the two McAbs(B1B6,B3D7)specifically recognized the epitope E(104)YGDLDART(112).Protein sequence alignment indicated that the sequences of E(104)YGDLDART(112)were conserved in all the reference strains.These results laid the foundation for the analysis of gB protein and facilitate the development of diagnostic method for PRV infection.4.Development of a blocking ELISA for detection of antibody to PRV with peroxidase-labelled monoclonal antibody against gB proteinA blocking ELISA method for detecting antibody against PRV was developed by using gB recombinant protein of PRV ZJO1 and HRP-lablled monoclonal antibody against gB protein.The conditions optimized for each step were as following:The optimal concentration of the coating antigen was 0.5?g·mL-1;serum samples detected were diluted with equivalent volume(1:1)and incubated for 1h at 37?;MAb-HRP(1:15000 dilution)is incubated for 0.5h at 37?.The cutoff of blocking ELISA was determined that the samples presenting a percentage inhibition of>28.67%were considered to be positive;samples with a calculated percentage inhibition of<18.27%were rated negative and those presenting a blocking effect between 18.27%and 28.67%were considered inconclusive.This method was proved to have no cross-reaction with positive serum of PRRSV,PCV2,CSFV and FMDV.The intra-assay and inter-assay variation coefficient were both less than 10%.The specificity and accuracy of the ELISA were 80.9%,96.4%.The rate of coincidence was 85.1%compared with the commercial IDEXX PRV gB antibody test kit.The detection of 535 clinical serum samples of piglets showed the positive rate for PRV antibody reached up to 61.87%.