Studies On The Interaction Between Rabbit Hemorrhagic Disease Virus VP60 Protein And Moesin Protein

Rabbit hemorrhagic disease (RHD) is an acute, potent, highly contagious disease of young rabbits more than three months, caused by rabbit hemorrhagic disease virus (RHDV). RHD was first reported in China in 1984 and outbroke in other countries throughout the world successively. So far, RHD has developed into a devastating epidemic of rabbit and seriously threaten the farming of rabbits. Great development has been achieved in RHDV research from RHD's first reported in 1984 so far. However, researchers nationally and internationally have not been able to find a cell line, in which RHDV can be stably propagated. As a result, the lack of a technology platform studying RHDV from a molecular level seriously retarded the research development of RHDV. Therefore, the pathogenesis mechanism of RHDV is still unclear.As is known to all, in the development of RHD, liver is the main organ of the virus multiplication, and liver's pathological changes are most severe in clinical pathological anatomy. While purifying RHDV in large scale, the dead rabbits'liver is an important source of the virus and researchers often extracted virus directly after homogenating livers. So, looking for the cell surface proteins interacted with RHDV VP60 from liver cell shows great importance, and research has shown that RHDV can infect primary cultured rabbit liver cells. VP60 protein is the major capsid protein of RHDV, and studies have shown that VP60 protein is the immune antigen of RHDV, playing an important role in the induction of immune response against viral infection, having a close relationship with virus'pathogenicity and immunogenicity.In this study, we built a eukaryotic expression library of rabbit liver cell by using SMART technology and screened the library by means of expression cloning. As a result, we get a protein which can interact with VP60 protein, Moesin protein. Expression cloning method is a classic method for screening and identifing functional gene; however, there may be still some false positive. To further determine the interaction between VP60 and Moesin, we used immunoprecipitation test and hemagglutination inhibition test to validate the interaction between Moesin and VP60 through in vitro. And confocal laser assay showed the same result.In our study, we first constructed the eukaryotic expression library of rabbit liver cell. The rabbit liver cDNA was cloned into the pEXP-Lib vector, and the identification results showed that distribution of inserts between 0.3 kb ~ 2.0kb,and recombination rate is about 86%. All results indicated that we obtained a high quality eukaryotic expression library. Then, using purified VP60 protein as a bait protein, we screened eukaryotic expression library by expression cloning, and screening results showed that 26 EST fragments have high homology with rabbit functional genes. By sequence analysis and literature review,we proceeded a further interaction verification and functional studies of Moesin.The fragment of Moesin gene 688nt ~ 2157nt was amplified from Rabbit liver cDNA, and was connected into the pET-30a and pEGFP-N1 vector respectively. The prokaryotic expression vector pET-30a-Msn was transformed into E.coli competent cells, and the soluble Moesin protein was induced and purified. Then immunoprecipitation test and HI test show the result that VP60 could interact with Moesin protein. Meanwhile, the eukaryotic expression vetor pEGFP-N1-Msn and pcDNA3.1-VP60 were co-transfected into HEK293T cells, and the interaction between VP60 and Moesin was verified again using confocal laser technology.In summary, we screened a protein gene, Moesin gene, from rabbit liver expression library, and expressed part fragment of Moesin protein successfully in vitro in this study. The discovery of interaction between VP60 and Moesin laid a foundation for further study of the pathogenesis of RHDV.