Preparation Of DuCV Nucleic Acid Probe And Subcellular Distribution Of Cap Capsid Protein

Duck circovirus (DuCV) is listed as a tentative member of the Genus circovirus. Ducks infected by DuCV exhibit disordered feather, retarded growth, reduced body weight. In the present study, we use the Baculovirus Expression Vector Systems to express and study the nuclear localization signal of the Cap gene of DuCVThe infectious clone of duck circovirus was labelled with digoxigenin as a DNA Probe. The hybridization assay of specificity showed that the DNA of the DuCV were positive, the other virus were negative. The sensitivity test showed that as low as 5 pg of DuCV's DNA could be detected by DIG-labeled probe. 1025 tissues samples from 196 diseased ducks which were collected in Shandong, Jiangsu and Sichuan were detected by the method of nucleic acid probe. The result is that the individual positive ratio of DuCV is 33.2%. The detection rates of cloacal bursa, liver and spleen were higher than other organs, and the detection rates were 52.3%,49.2%,38.5%.The whole Cap gene was amplified by polymerase chain reaction (PCR) and then cloned into the baculovirus donor vector pFastBacTM HTB according to the right open reading frame (ORF). The recombinant plasmids pF-CP DNA were transformed into E.coli competent cells DH10Bac containing baculovirus shuttle vector. The recombinant bacmid (rBacmid-CP) was screened and verified by PCR. The rBacmid-CP was transfected into Sf9 insect cells and the recombinant baculovirus was obtained and confirmed by western blot and immunofluorescence assay with the antiserum was collected from the mice which were immunized with the Cap of prokaryotic expression. The recombinant Cap would be useful resource for studying on the function of Cap.36 alkaline amino acids present in N-termination of Cap protein of Duck circovirus (DuCV) FJ0601. After being analyzed by PROSITE, two bipartite nuclear localization signals were found in this region (separately be situated at 2～17 and 21～36 of N-termination of Cap protein). For the further research of Cap protein, a series of plasmids related to Cap protein were constructed and research about nuclear localization signal were carried out. Firstly, EGFP was inserted into pFastBacTM HTB to construct the recombinant plasmid (pF-EGFP). Then, pF-EGFP-CP, pF-EGFP-CP-Δ17N(missing 2-17 amino acids from N-termination)and pF-EGFP-CP-Δ36N(missing 2-36 amino acids from N-termination)were constructed and transfected Sf9 cells. Results indicate that protein of pF-EGFP-CP and pF-EGFP-CP-Δ17N locate in the nucleus while protein of pF-EGFP-CP-Δ36N locate in cytoplasm. Coincident with prediction results of PROSITE, it indicates that nuclear localization signal locates in 21～36 amino acids of N-termination.