Development Of Indirect Elisa For Detecting Antibodies Against GB Protein Of Porcine Cytomegalovirus And Preparation Of Its Monoclonal Antibodies

PCMV is an enveloped virus containing a linear double-stranded DNA genome.Infected pigs present with clinical symptoms of death of embryo,rhinitis, pneumonia,dysplasia,and recovered pigs develop lifelong latent infection.Reported major proteins of PCMV contain major capsid protein(MCP),DNA Polymerase Loci protein(DPOL) and Glycoprotein B(gB). PCMV gB protein is an important transmembrane glycoprotein which plays a major role in the adhesion and fusion of the virus onto the cell membrane of the host cells when the virus enters cells or transfers between cells.As the major protective antigen,gB protein can induce specific immune response.In this study, recombinant gB protein was expressed and purified.Using purified recombinant gB protein,an indirect ELISA was developed for detecting antibody to PCMV and used for epidemiological survey of PCMV.Meanwhile,monoclonal antibodies were prepared which laid a foundation for the further study of diagnosis and pathogenic mechanism of PCMV.The main contents were as following:1.Prokaryotic expression and antigenic analysis of gB proteinPCMV gB gene was amplified by PCR and cloned into a prokaryotic expression vector pET-32a.PCR and restriction endonuclease analysis and sequencing were directed first to identified recombinant plasmid pET32a-gB.Protein was then expressed in E.coli BL21(DE3) cells by induced with IPTG.SDS-PAGE analysis reviewed expressed protein was about40KDa.Correctness and antigenicity of expressed target protein were identified by Western-blotting analysis.All results indicated that the expressed recombinant gB fusion protein has antigenicity.2.Evelopment and application of an indirect ELISA for detecting antibodies against gB protein of PCMVAn indirect ELISA method was developed using purified recombinant gB protein as coating antigen after optimization of reaction conditions. The optimal antigen concentration and serum samples dilution were set at0.4μg/mL and1:100. The coated time was2h at 37℃then overnight at4℃.The plates were blocked by5%skim milk incubated at37℃for2h. The conjugate was diluted at1:10000,incubated at37℃for0.5h.It was judged as positive when the cutoff value OD450nm≥0.354,as negative when OD450nm≤0.295, and as suspicious between0.354and0.295.It did not react with the swine serum antibodies to swine fever,pseudorabies,porcine reproductive and respiratory syndrome, porcine circovirus type2and Haemophilus parasuis.And it had good inter-and intra-batch reproducibility.Total259clinical serum samples obtained from Yangzhou, Jurong, Shuyang were detected.,and the positive rate was56.76%. Developed ELISA could be used for PCMV epidemiological surveys and diagnosis in the future.3.Preparation of monoclonal antibodies against gB protein of porcine cytomegalo virusThe monoclonal antibodies (MAbs) were prepared by fusing mouse myloma cells (SP2/0) with spleen cells from BALB/c mice immunized with purified recombinant gB protein of Porcine cytomegalovirus. Three hybridoma cell lines secreting MAbs against gB protein were screened by indirect enzyme-linked immunosorbent assay (ELISA) and named as6C2、6H9and8B5,respectively. The titers in supernatant and asicite were1:800～1:3200and1:4×106～1:8×106respectively. The isotypes of6C2and6H9and8B5belong to Ig2b/κ;1E7,3C9,3F6,3Flland5G10belong to IgGl/κ.Western-blot results showed that the two of eight MAbs(6C2and6H9) reacted specifically with recombinant gB protein expressed in Prokaryotic and eukaryotic cell.In a summary, an indirect ELISA was developed for detecting PCMV antibodies and at the same time,monoclonal antibodies were prepared of gB protein, which laid a foundation for the epidemiological surveys and diagnosis of PCMV and development of PCMV quick-diagnosis kit.