| Circovirus, mainly do harm to the immune system,can infect various animals and cause great losses to the cultivation industry,which attracted worldwide attention. Duck circovirus(DuCV) reported by Hattermann for the first time is the smallest virus founded in the infected ducks up to now, and blongs to the family Circoviridae,genus Circovirus. It is a single-stranded DNA virus with a circular genome of approximately 1.9 kb,non-enveloped, spherical or icosahedral symmetry, about 15 nm in diameter. The genome contains two major open reading frames (ORF):ORFV1 encodes the replicase (Rep)proteins and ORFC1 encodes the capsid (Cap) protein.Till now the pathogenic characteristics and mechanism of Duck circovirus are still unclear.But DuCV prevalence has been reported at home and abroad repeatedly.Thus we can get the conclusion that Duck circovirus prevalents widely, which needs more attention.As Cap protein is the major antigen region where the protective antibody produced, it has huge potential values for wide application. In order to obtain the full-length cap protein, virus genome was extracted from duck bursa of fabricius collected from east China. Afteer primers designed based on the published duck circovirus (EU022375) sequence(774 bp), the full-length of DuCV-cap gene were amplified by PCR,then the fragments were inserted into pET32a(+) vector to construct the recombinant expression vector so as to express DuCV Cap protein. It has been reported that the whole cap gene could not be expressed in E.coli system for the accumulation of arginine residues in the amino-terminal region, and Shao-Ning Liu had espressed the truncated DuCV-Cap protein of which the first 36 amino acids were deleted.While in this study, we have expressed the full-length Cap protein successfully, for its antigenic region is longer than the truncated one,so it will have better antigenicity.In order to investigate the infection circumstances and the effect of duck circovirus in east China, added that there is no commercialized ELISA kit for duck circovirus antibody diagnosis at present, we developed an indirect ELISA method based on the purified recombinant fusion protein to detect DuCV antibody, ultimately determined that the concentration of antigen was 12.5μg/ml, the best proportion of diluting duck serum was 1:80. When we estimated the values of the samples, we considered OD450nm≥0.40 as positive, while OD450 nm<0.40 was determined to be negative.As this method was appraised to be specific and sensitive after being checked.321 serum samples (different ages,4 varieties) collected from 19 farms in east China were detected with this method, the total DuCV antibody positive rate was 45.8%, among which it was 26.7% in Jiangsu Province, and 60.9% in Shandong Province.The analyzed data showed that with the increase of the age,DuCV antibody positive rate increased. Meanwhile, DuCV infection of different species are also different, indicating that there are some certain links between DuCV infection and specific species.Ducks are the main host of avian flu virus and important vector for its circulation, so avian flu vaccine is critical to ducks, but the antibody after vaccineation often showed instability. So we surveied the avian influenza (H5 and H9) antibody titers on the same batch of Serum to uncover the possible reason of this phenomenon. Statistics of the failed antibody titers of avian influenza in DuCV positive and negative samples respectively (the failed antibody titers of avian influenza in DuCV positive samples vs. the failed antibody titers of avian influenza in DuCV negative samples)showed that:H5subtype,Shandong (16.7% vs.9.8%), Jiangsu (57% vs.36.3%); H9subtype, Shandong (47.2% vs.19.7%), Jiangsu (75% vs.69%). From the results we can concluded that the failed antibody titers of avian influenza in DuCV positive samples is higher than those in DuCV negative ones with significant differences.This indicated that the infection of DuCV had inhibited the production of normal antibody after immunization, therefore we can provide evidence for the harm of duck circovirus infection.In order to do the further research about DuCV, we also invented the monoclonal antibodies with the purified DuCV recombinant Cap protein as immunogen. Balb/c mice were immunized according to the conventional methods, Spleen cells of the immunized mice were fused with SP2/0 cell (the fusion rate was more than 95%),after being screened by indirect ELISA method, three hybridoma cell strains against DuCV-Cap fusion protein were obtained and named as 1G5B11,2E6C1 and 3E9C7 respectively.The ELISA titer of culture supernatant were 1:1024,1:512,1:1024,and those of ascites were all higher than 105.The three McAb all belonged to IgG1 subgroup after being detected by the Ig subtype kit. Then the sub-Cap protein was used as antigen in Western blot analysis, results showed that three monoclonal antibody could be identified by the C-terminal-truncated Cap protein, but could not be indentified by either the segment C1(154-258 aa) or C2 (53-209 aa), which indicated that the specific epitopes located approximately in the fomer 52 amino acids within the N-terminal of the Cap protein. For there has been no correlated reports about the epitopes on DuCV Cap protein, this research will provide some foundations for the development of genetic engineering vaccines on DuCV... |
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