| Monoclonal antibody technology was created by G. Kohler and C. Milstein in 1975. Hybridomas cells producing McAb are prepared by cell fusion technique and inherit the characteristics of parental cells, which can secrete antibodies and infinite reproduction in vitro. It has become an indispensable tool which is widely used in immunology, cytobiology, oncology, medical and microbiology because of strong specificity, good reproducibility and high purity of monoclonal antibody (McAb). It was mainly applied for the detection of virus, study of antigenic epitope and function of protein in veterinary science.The diseases associated with porcine circovirus (PCV) infection are called porcine circovirus disease (PCVD), which mainly include postweaning multisystemic wasting syndrome (PMWS), porcine dermatitis and nephropathy syndrome (PDNS), porcine respiratory disease complex (PRDC) and PCV2-associated reproductive failure. Wide prevalence and serious damage of PCVD has made a great economic impact on the swine industry.Porcine circovirus (PCV) belongs to the family circoviridae, genus cirvovirus, and is a small (17 nm), nonenveloped, icosahedral virus containing a circular, single-stranded DNA genome. According to genetic studies, PCV was divided into PCV1 and PCV2 genotypes and the length of DNA genome is 1,759 bp for PCV1 and 1,768 bp or 1,767 bp for PCV2. The genomic DNA of PCV2 consists of two major open reading frames, ORF1 and ORF2. ORF1 of PCV2 is 945bp in length encoding a replication-associated protein and ORF2 of PCV2 is 705 bp in length encoding a major capsid protein, which can stimulate body to produce protective antibody because of its good immunogenicity.The Cap protein with deleted nuclear localization signal peptide was expressed in recombinant E. coli M15. The expressed protein was purified through Ni-NTA agarose metal affinity resin column. UV-spectrophotometer showed the purified protein was 1.2mg/mL . BALB/c mice aged six weeks were immunized three times with purified protein which was mixed with freund's adjuvant during a two-week interval, 100μg pur mouse. The titers of serum after third immunization was up to 1.0×10~4, which indicated the mice could be used for cell fusion. Hybridoma cells were made by fusing the spleen cells with SP2/0 myeloma cells and were screened by testing the hybridoma culture supernatant. Three positive hybridoma cell lines named 2C3, 2F3 and 1E10 were obtained.The number of chromosomes in hybridoma cells treated with colchicine was analyzed after fixation and staining. The result showed that the number of chromosomes was among 95 to 103, confirming the realness of hybridoma cells. The hybridoma cells were injected into mice abdominal cavity to prepare ascites. The titers of ascites were 1.0×105 by ELISA. 2C3 and 2F3 McAbs belong to IgG1 isotype while 1E10 belonge to IgG2a isotype, and all the light chain of three McAbs isκchain. The result of western-blot assay showed that all the McAbs can specially react with Cap protein. PK-15 cells was infected with PCV2 and fixed by acetone, then the IFA was carried out using the prepared McAbs. The result showed that all of the McAbs reacted with PCV2.293T cells were transfected with recombinant plasmid pCA-Cap containing full length of Cap gene (Cap) and recombinant plasmid pCA-TCR containing Cap gene with deleted nuclear localization signal (dCap) respectively. 293T cells were fixed by paraformaldehyde after 72h. Then the following steps were carried out: McAb 2F3 for 2h incubation, FITC-IgG for 1h incubation, PI (Propidium iodide) for 5 mins, observation under confocal microscope. The result showed that the Cap protein was mainly located in nucleus, while the dCap protein was located in cytoplasm.Three McAbs against PCV2 Cap protein were successfully prepared and subcellular localization application of PCV2 Cap was carried out using McAb 2F3. The prepared McAbs laid the foundation for establishing the diagnostic method and studying the antigenic epitope for PCV2.
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