Detection Of Virus In Immune Tissue And Study Of Immunosuppression On Goose Infected By GPV

Goose parvovirus (GPV) is the etiological agent of Gosling plague (GP), an acute, contagious, and fatal disease, which is also known as Derzsy's disease. It was first described as a clinical entity by Fang. GPV mainly infect 4-day-old to 20-day-old goslings and Muscovy duckings. It has a high mortality,which mostly threaten the goose industry. Scholars carried out on a lot of research about GP and have made some progress.At present, the studies about pathogenesis of goose plauge are seldom carried out. In this study, Coding sequence of VP3 gene was cloned by the methods of molecular biology. VP3 was expressed in prokaryotic system by genetic engineering technology. The polyclonal antibody was prepared using expressed fusion protein. The distribution of GPV in vivo was detected by the applications of PCR and immunohistochemistry. The expression levels of IL-8,IL-18,IFN-alpha,IFN-gamma mRNA and PPAR-gamma mRNA in spleen, thymus, and bursa of Fabricius of gosling which were infected by GPV were determined by real-time PCR. The main results were showed as follows:1 According to the published of GPV B strain genome nucleotide sequence in GenBank and a pair of specific primers were designed with Oligo6.0 and Primer Premier 5.0. Full-length VP3 gene was amplified by ploymerase chain reaction(PCR). The gene VP3 was cloned into pMD18-T vector and sequenced. VP3 gene was cloned into the multiple cloning sites of prokaryotic expression vector pGEX-6P-1. The recombinant plasmid pGEX-6P-VP3 was constructed and transformed to Rosetta, positive bacterium strain was induced by IPTG. SDS-PAGE reveals that the VP3 gene was expressed in Rosetta with high efficiency.The weight of protein is 83KD,which is fussed with GST.2 The protein was used to immunize female Balb/c mice. After three rounds of immunizations, antiserum was collected and used to detect the specificity of recombinant protein. ELISA showed that the titer of antiserum was above 1:25 600. Moreover, antiserum reacted specifically with purified recombinant protein in Western-blot..3 The content of GPV in spleen, thymus, bursa of Fabricius of gosling was deteted by PCR and immunohistochemistry.4 GPV infection can cause damage to the immune structure.The expression level of IL-8,IL-18,IFN-α,IFN-γmRNA in spleen, thymus, bursa of Fabricius was deteced by real-time PCR. The result indicated that 8 days after the viral infection immune tissue IFN-α, IFN-γ, IL-8, IL-18 mRNA lower expression levels, it suggested GPV infection caused immune suppression of goslings.5 Through the detection of the expression level of PPAR-γmRNA in spleen, thymus, bursa of Fabricius, it was demonstrated that the gosling PPAR-γgene was also expressed in thymus and bursa of fabricius. The results also indicated that GPV infection could alter the mRNA levels of PPAR-γgene in spleen, thymus, bursa of Fabricius , and the magnitude and direction of change in PPAR-γgene expression differed with the different stages of infection and also varied by tissue. This indicated that PPAR-γgene played an important role in the immune regulation of the immune system in gosling after GPV infection.