Research On Interactions Between Differentially Expressed Proteins In Rabbit Liver Tissue And Rabbit Haemorrhagic Disease Virus

Rabbit haemorrhagic disease virus (RHDV), as a contagious pathogen, is a calicivirus of the genus lagovirus that causes acute death of adult rabbits. Adult rabbits showed notable clinical symptoms at30hours post-inoculation (PI), and ninety percent rabbits died from48h to72h PI. However, rabbits younger than45days old were not susceptible to RHDV and did not show clinical signs of the disease. The difference of susceptibility to RHDV may closely associated with the differentially expressed function proteins in different development stages. In addition, the liver, the place of virus proliferation, do not express the attachment factor HBGA of RHDV. Therefore, screening of the differentially expressed proteins between adult and young rabbits and investigating the relevance with RHDV have significant meaning for clarifying the pathogenesis of RHDV.In this study, we applied two-dimensional fluorescence difference gel electrophoresis (2-D DIGE) technology to separate the differentially expressed proteins of liver tissues between adult and young rabbits, and identified these proteins by MALDI-TOF-TOF MS technology. The results showed that46protein spots were significantly different, and among them28protein spots were up-regulated,18down-regulated in the liver tissue of adult rabbits. Then Gene Ontology (GO) and protein-protein interactions of differentially expressed proteins were analyzed by bioinformation. Moreover, three differentially expressed proteins:fatty acid binding protein7(FABP7), aldo-keto reductase family1, member C1(AK1C1) and adenosylhomocysteinase (AHCY) were selected in random to test their expression at mRNA level by real-time PCR. The results were consisted with DIGE.In order to identify the direct interactions between differentially expressed proteins among adult and young rabbits’ liver and viral proteins encoded by gRNA, yeast two-hybrid system was used. Total RNA of liver tissue and viral RNA of HYD strain were extracted by Trizol method and reverse transcribed into cDNA, viral structural protein VP60, minor structural protein VP10and seven nonstructural proteins p16, P23,2C-like protein, P29, VPg,3C-like protease, RdRp and46differentially expressed proteins were amplified by PCR, and then constructed to pGBKT7and pGADT7vector respectively. Recombinant plasmid pGBKT7-X and pGADT7-Y were tested to be no autoactivation and toxicity and then co-transformation into AH109to identify the interactions. The results showed that VP60and NmrA-like protein, VP60and Nitrilase family, member2(NIT2) were positive interactions. NmrA-like protein was related to the activity of GATA transcription factor and NIT2was reported to involve in regulation of cell growth.In summary,2-D DIGE coupled with MALDI-TOF-TOF MS technologies successfully obtained differentially expressed proteins in liver tissue between adult and young rabbits, and yeast two-hybrid screened the proteins that may interact with RHDV, laying the foundation for clarifying the mechanism of RHDV in cells’ transcriptional regulation and infection.