Proteomics Analysis Of Rabbit Liver After Infeciton By Rabbit Hemorrhagic Disease Virus

Rabbit haemorrhagic disease virus (RHDV) is a calicivirus of the genus Lagovirus that causesrabbit haemorrhagic disease (RHD) in adult rabbits. First described in China in1984, the virus rapidlyspread worldwide and is nowadays considered as endemic in several countries. The liver, lung andspleen are the primary target tissues of RHDV. The major histopathological lesions found at necropsyare acute hepatitis due to liver cell loss as the result of RHDV-induced apoptosis, and splenomegaly.Therefore, to study the differences of protein expression profiles during the process of RHDV infectioncould help us to further elucidate the molecular mechanisms of pathogenicity.In our study, we used proteomics techniques to study the global protein expression change profilesof rabbit liver following RHDV infection in vivo. Total liver proteins were extracted from mature rabbitchallenged by RHDV and the mock-infected. Differentially expressed proteins were separated bytwo-dimensional gel electrophoresis, analyzed by mass spectrometry (MALDI–TOF-TOF) and proteindatabase searching. Forty-nine significantly different protein expression spots were screened, amongwhich fifteen protein spots were identified to be up-regulated and thirty-four down-regulated in RHDVinfection. We presume that five of them were involved with the infection of RHDV, includingalpha-1-antitrypsin（A1AT），serum albumin precursor(PA), laminin receptor，cytochrome P-450，fibrinogen and heterogeneous nuclear ribonucleoprotein（hnRNP）. In addition, the transcriptional levelsof3genes (A1AT，PA and hnRNP) were verified by real time PCR, two of which was consistent withthe proteomic result. Next we also proceeded with further interaction verification and functional studiesof Α1AT which is one of the significantly different protein expression spots.To prokaryotic and eukaryotic express the fragment of A1AT gene, the fragment were amplified byPCR with primers designed according to the sequence of A1AT gene and inserted into pET-30a vectorfor expression in E.coli and pEGFP-N1vector for confocal assay respectively. The interaction betweenA1AT protein and VP60could be verified by immunoprecipitation test and HI test. The GST-polldowntest also indicated that VP60could interact with A1AT protein. Finally, with the help of confocal lasertechnology we verified the interaction between A1AT and VP60after pEGFP-N1-Msn andpcDNA3.1-VP60co-transfected into HEK293T cells.In summary, the proteomics analysis of rabbit Liver provides valuable insights into the interactionsof RHDV with its host and the investigations in pathogenesis of RHDV. Meanwhile, the results of theinteraction between VP60and A1AT in our studies would be facilitated further study of thepathogenesis of RHDV and Host-virus co-evolution.