Preparation Of Monoclonal Antibodies Of Dhv-ⅰand Development Of Colloidal Gold Strip For Detection Of The Virus

Duck hepatitis is a kind of highly contagious and lethal infectious disease which caused by duck hepatitis virus. DHV can infect young duck mainly within the 3-week-old ducklings. In this study, VP1 protein of DHV-Ⅰwere expressed by Prokaryotic expression system, the monoclonal antibodies of DHV-Ⅰwere prepared and the colloidal gold strip for detection of the virus were developed.The VP1 gene of DHV-Ⅰwas amplified by RT-PCR, and the PCR products were cloned into pET-32a(+) vector directionally, then the prokaryotic expression recombinant plasmid was transformed into Rosetta(DE3)PLys. The fusion protein expressed in E. coli induced by IPTG were 47 kDa, and the protein could react with positive serum by western blot, demonstrating that it had good immunogenicity.The Balb/c mice were immunized with purified antigen of DHV-Ⅰ, and indirect enzyme linked immunosorbent assay (iELISA) used to screen hybridoma cells was developed. The best reaction conditions of iELISA were that the purified antigen diluted as 1:800 and coated in 4℃for one night, the negative and positive serum were diluted as 1:4000, the temperature of the reaction was 37℃and the reaction time was 90min. The spleen cells from immunized mice were fused with SP2/0 and 21 potive hybridoma cells were obtained by iELISA. Limiting dilution method was performed to subclone positive hybridoma cells, and 5 potive hybridoma cells that could secret McAbs stably were obtained finally after 3-4 subcloning, named as 1E10, 4F8, 4E6, 5G4, 5E11. The stability, chromosome number, subtype, titers of the five McAbs in the ascites and specificity were identified. The ability of secreting McAbs of the hybridoma cells were stable after serial passaging for 20 times,whick indicated that the stability of the five hybridoma cells were good. The chromosome numbers of the five hybridoma cells were between 100 and 110, which were according with the chromosome number of the hybridoma cells. The subtype of 1E10, 4F8, 4E6, 5G4 and 5E11 were IgG2b, IgM, IgG1, IgM and IgG1 respectivly, and the titers of the McAbs in the ascites were 1: 400, 1: 8000, 1: 8000, 1: 32000 and 1: 16000. All the five McAbs were specific for DHV-Ⅰ.The McAbs were purified by ammonium sulfate precipitation method, and were labeled successfully by colloidal gold. The combination was optimized with the conditions of the check line and the contrast line sprinkled 1mg/ml and 1:10 diluted protein when makes the strip, meanwhile 2 lines sprinkled 1μL/cm. The glass fibre of Ahlstrom 8964 and GRADE7589 were used as chromatographic materials to develope colloidal gold strip. Using diluting buffer as negative control and DHV-Ⅰdiluted with diluting buffer as positive control, the negative control only appeared a contrast line while the positive control appeared a check line and a contrast line on the colloidal gold strip. Clinical test results indicated that the colloidal gold strip were specific and sensitive for detecting DHV-Ⅰ.