| Duck viral hepatitis(DVH)is an acute,lethal infectious disease with a high mortality rate of ducklings caused by duck hepatitis virus(DHV).At present,the prevalent DVH pathogen in China is duck hepatitis A virus(DHAV),the virus has three serotypes: DHAV-1,DHAV-2,and DHAV-3.Mainly popular strains in China are DHAV-1 and DHAV-3,because there are no vaccines successfully developed,and there are reports of viral variants in the word,so DVH can not be effectively controlled,which bring a huge loss to the duck industry in China.In this study,we used the His labeled pET-30a-VP1-1-221 aa plasmid preserved in the laboratory as a template to design primers without His label pET-30a-VP1-1-221 aa,and use the prokaryotic expression system to prepare the protein without His tag VP1-1-221 aa as immunogen,immunization of BALB/c mice,fusion of myeloma SP2/0 cells with spleen cells of immunized mice.The His-tagged VP1-1-221 aa truncated protein as the detection source,and the established I-ELISA method was used for detection.Two(2E11,4F3)positive hybridoma cells which are capable of secreting monoclonal antibodies anti-DHAV-3 VP1 protein stably were obtained after five times subcloning screen.The results of subtype identification of monoclonal antibodies showed that the 2E11 strain was IgG1 type,4F3 strain of IgG2 a type.The ascites of monoclonal antibodies were prepared and the titer of ascites was 1:10240.In order to identified the antigen dominant region that were recognized by two strains monoclonal antibodies,the DHAV-3 VP1(1-221aa)protein was expressed in two rounds,the recombinant expression plasmid was constructed,and transformed into the competent cells,then the fusion protein was induced by IPTG which were identified by SDS-PAGE,finally the supernatants of two monoclonal antibodies were used for Western blot analysis.According to the Western blot results and the strategy of truncated protein,the results showed that the VP1-1-e truncated protein reacted specifically with 2E11 and 4F3 monoclonal antibodies,that is to say,the 80aa-110 aa was the antigen dominant region of DHAV-3-VP1-1-221 aa.The ultracentrifuged recombinant virus rDEV-?Us10-3-VP1 and the duck embryo allantoic fluid of DHAV-1 and DHAV-3,were detected by Western blot used the ascites of two strains monoclonal antibodies.The result indicated that all of them only recognized DHAV-3 VP1 protein,but could not recognized DHAV-1 VP1 protein.IFA results showed that two strains monoclonal antibodies specifically reacted with the recombinant virus rDEV-?Us10-3-VP1 with green fluorescence,whereas the no specific fluorescence generated with the parental virus DEV C-KCE.The results showed that two monoclonal antibodies recognize the DHAV-3 VP1 protein as same as the result of Western blot.The neutralization activity of 2E11 monoclonal antibody was determined by the method of keeping the constant titer of the virus and diluting the supernatant of hybridoma cells.The results showed that 2E11 monoclonal antibody had the neutralizing activity and it can protected the 50% duck embryos far away from the death when it was diluted 1:12.6. |
