| Duck viral hepatitis is an acute and fatal disease of young ducklings, especially under 4 weeks, caused by duck hepatitis A virus. There is an enormous damage to the duck industries because of its higher mortality and severe infection. In order to developt the vitro model of DHAV-1 and research the immunogenicity of VPO protein of DHAV-1, a serial studies are done in this paper, including the adaptivation of DHAV-1 in DEF and the structural protein VPO. They are five parts in this thesis.In the first part, duck embryo fibroblast(DEF) was inoculated with allantoic fluid of the wild strain DHAV-1-X isolated and saved in our laboratory to passage the virus. Three passages later, a weak CPE was observed in cells and after 8 times, infected DEF represented obvious CPE. A series of tests, including RT-PCR, immunofluorescence and inoculation on the duck embryo, were done to demonstrate that DHAV-1-X could proliferate excellently in DEF. The 14th passage virus was choosed to detect the propagation of the virus by using real-time PCR with higher detection sensitivity and specificity, which suggested that the virus began to entry the stage of active replication after 12h post-infection, and reached the peak at 48h, and continued to release the virus to supernatant; the content of the virus maintained at a certain stage at 48h-96h, which suggested that the virus titer tended to be stable; the cells began to disintegrate and the content decreased after 96h.In the second part, the gene sequence of VPO (VPO-F) and the partial sequence of VPO (VPO-P) of DHAV-1-X were correctly cloned into pET-32a (+) vector and pGEX-4T-1 vector, respectively. The reeombinant expression plasmid pET-32a (+) /VPO-F and pGEX-4T-1/VPO-P was expressed in E.coli expression system. The fusion protein VPO-F and VPO-P were expressed in E.coliBL2\and confirmed by SDS-PAGE. The protein of VPO-F and VPO-P were purified and collected, respectively, which would be identified in serum against DHAV-1.In the third part, the serum of rabbit-anti-VPO-P was prepared by immunizing the rabbits with the pruified VPO-P. The antibody level of the serum was detected 1:16 through agar gel immunodiffusion. At the same time, the neutralization titer of the serum was 1:146 detecting by chicken embryo neutralization test, and the serum could be recognized the DHAV-1 successfully by indirect immunofluorescence technique.In the fourth part, VPO-F ELISA and VPO-P ELISA was established based on the two recombinant proteins, respectively. The results showed that the best condition of VPO-F was coating with 1.67μg/ml at 37℃ for 1h to 4℃ overnight, and blocking 0.5h with 5% gelatin, then inculabted with 1:160 diluent of serumm for 1h, following with 1:400 diluent of HRP-goat-anti-duck IgG for 1h. The value of OD450/OD630 was detected after combined with TMB for 10min and the positive threshold is 0.375. The results of VPO-P also showed that the best condition was coating with 1.67μg/ml at 37℃ for 2h, and blocking 2h with 5% gelatin, then inculabted 1h with 1:80 diluent of serumm, following with 1:400 diluent of HRP-goat-anti-duck IgG for 1h. The value of OD450/OD630 was detected after combined with TMB for 5min and the positive threshold is 0.317. Both ELISA methods was specific and repeatability, and the rate of coincidence of the ELISA methods and DHAV-1-ELISA was 90% and 93.3%, respectively.The last part, to detect the antibody level and the level of CD4, CD8, IL-4 and IFN-y, one-day ducklings were vaccinated with VPO-F and VPO-P and were bleedd at different ages. The level of antibodies reached a peak at 15 old age and after that the level began to decline, and the immune effect of VPO-F was better than that of VPO-P. The content of cytokines reached a peak at 7 old age and the level was similar to the level of antibodies. |
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