| Duck plague is an acute, septic contagion, caused by duck plague virus, with the characteristics of head and neck swelling, the mucosa of esophageal and cloacal bleeding and yellowish-white ulcer, head and neck skin having a yellowish-white gelatin sample. Once it broke out, with high morbidity and high mortality, it would cause serious harm to the duck industry. Immunization with DPV vaccine have taken some effect, but in recent years, the effect is not very well. Whether is the procedure of vaccine or the variation of DPV gene, it is important to find the reason. Now there are many methods to detect DPV, for example,PCR, ELISA, indirect immunofluorescence, Microneutralization test and so on, to lay a good foundation for the diagnosis and treatment of the disease. But these methods take long time with low sensitivity and they are not useful for detecting duck plague virus. So it is very urgent to establish a more rapid and sensitive detecting method. Immunochromatography colloidal gold strip is a sensitive method to make rapid detection for DPV. Monoclonal antibody can supply a good tool for making rapid detections for DPV, with stable properties and high specificity. As one of the highly conserved proteins, protein B can supply a good material for establishing of DPV detecting method, with good immunogenicity and high specificity and conservatism.We have amplified the encoding sequences of DPV gB and gC gene from various regions in recent years, to observe the variations from the molecular level. We have prepared the monoclonal antibodies against DPV, to supply a good tool for immunochromatography colloidal gold strip.We first designed the specific primers of DPV gB and gC genes. After handling the virus materials from various regions in resent years, we had isolated DPV by duck embryo allantois membrane inoculation. Then we extracted the viral DNA as a template to amplify the aim gene. It was connected to the pMD-18 T vector after that. After sequencing the positive bacteria liquid, identified by PCR, we compared them with the DPV sequences published on NCBI. At the same time, we compared them with each other. The results showed that the homology of the sequences of the nine DPV strains was between 99% and 100%. It can indicate that the DPV was not verified, we can preliminary make a conclusion that it may bethe vaccine problems.Next, we have chosen the main antigenic domain of DPV by the analysis of the Protean Biology software to design a pair of primer to amplify the aim gene by PCR. Then the fragment was inserted into prokaryotic expression vector pET-28 a to construct recombinant plasmid.Then it was transformed into Rosetta for expression. During the experiment, we have groped the concentration of the IPTG and the induction time. After purifying, the aim protein has been tested its concentration and has been analyzed and identified by western blot.Four hybridoma cell lines designated as A8D9, E6C3, H11F8, H6A10, stably secreting monoclonal antibody against gB protein of DPV were generated by fusing SP2/0 myeloma cells with splenocytes from the immunized mice, which use the gB protein of DPV, expressed and purified with prokaryotic, as the antigen.The titres of ascitic fluid was1:103, 1:103, 1:105,1:103, respectively by indirect ELISA and the immunoglobulin subtype of the monoclonal antibodies was IgG2 b, IgG2 a, IgG2 b, IgG1,with the light chain of kappa. The result of western blot showed the four monoclonal antibodies were able to specifically recognize gB protein of DPV. The result of IFA showed the four monoclonal antibodies were specific to DPV.At last, the monoclonal antibodies-based colloidal gold immunochromatography strip was developed for the detection of DPV. The purified DPV-gB monoclonal antibody, named H6F6, was labeled with colloidal gold, with the appropriate pH between 8.0 and 8.5 and the concentration was 15 times dilution. The purified A8D7 monoclonal antibody, with the concentration of twice dilution, and the goat anti-mouse immunoglobulin G(IgG) antibody,with the concentration of ten times dilution, were blotted on nitrocellulose membrane as test line and control line respectively. The detection results indicated that the strip was specific to DPV and had no cross reaction with DRVã€EDS-76Vã€AIV-H9N2ã€TMUV. The detection limits of DPV were 50 times dilution. 38 clinical suspected samples were simultaneously detected by immunochromatography strip and PCR while the results showed 91.6 % accuracy between them.The monoclonal antibodies-based colloidal gold strip was highly specific and sensitive and more convenient for the clinical diagnosis of DPV. |
