Construction Of A Plasmid-based Infectious Clone And Study Of The Interaction Between Structural Protein Vp60 And Vp10 Of Rabbit Hemorrhagic Disease Virus

【Objective】Currently, the pathogenesis of the Rabbit hemorrhagic disease virus (RHDV), structure and function of Genome, virus assembly mechanism and so on, that is not fully understood. In order to provide a good technical platform for going into RHDV, and to provide clues for studying the assembly mechanism of RHDV. This study optimized the Reverse genetic operating system of RHDV, and build the infectious cloning based on Plasmid transfection,and proclaim that there was mutual effect among the structural proteins of RHDV.【Methods】(1)Add T7 promoter core sequence in the 5' end of RHDV cDNA sequence by PCR reaction, and add Ribozyme core sequence which has its own cutting function in the 3 'end.Assenble the different fragment of cDNA of the RHDV genome into the pTVT transcription carrier, constructs the recombinant plasmid pTVT-RHDV which contains the full-length cDNA sequence of RHDV. And transfect the BHK cell which can Express T7RNA polymerases stably,infect MA104 cells with the cell culture harvest, to get the rescued RHDV.Identify the rescued virus by RT-PCR and IFA.(2)By Check Mate? Mammalian Two-Hybrid System, the recombinant plasmid containing VP60 and VP10 was build respectively, and then transfect the Hela cell, identify whether there is ineraction between VP60 and VP10 by detecting the expression of the Reporter gene(Luciferase)and then check it futher with Confocal immune and immune precipitation and so on.【Results:】(1)From the cell culture, the tag sequence with the specific purpose of the molecular was successfully amplified, the antigen of RHDV was highly expressed in the MA104 cell, the results of step growth curve of the test showed rescued virus had similar growth kinetics characteristics with the wild type virus. It showed that RHDV was saved successfully.(2)Luciferase was expressed highly,after Hela cell was transfected,there are some overlap about the Specific fluorescence between VP60,VP10 and antibody, the result of Immunoprecipitation experiment shows there are immune precipitation compound between the VP60 and VP10. The result showes, there are specific interactions between the RHDV's capsid protein VP60 and the major structural protein VP10. But when VP60 was cut into 2 sections, VP60N and VP60C, the interaction between them and VP10 was seriously weakened.【Conclusion】(1)An easy operation, high efficiency save RHDV infectious clone was built successfully(.2)Proclaimed there was specific interactions between the RHDV's capsid protein VP60 and the major structural protein VP10, and the interaction is highly dependent on the structure of the capsid protein.