Immunogenicity Of VP6and NS38, And Screening Of The Proteins That Interacted With VP6and NS38in Grass Carp Reovirus Strain096

The incidence of Grass Carp Hemorrhage is high and wide, but there is not aneffective drug to prevent and treat it, and it is still a bottleneck in the development of thegrass carp. The major pathogen of grass carp hemorrhage is grass carp reovirus (GCRV),which infects grass carp fingerlings and has a serious impact on the development ofChina’s freshwater fisheries. The practice has proved that immunization is one of the mosteffective method to prevent the disease. The antigenic differences exist among differentstrains, and there is no cross-antigen. Studies have shown that the VP6protein couldinduce more antibody than NS38protein, though both of them can induce the generation ofantibody. The further understanding the immunogenicity of VP6and NS38proteins inGCRV will have more important value for preventing grass carp hemorrhage. Virusesinvade cells of the host and breed themselves, resulting in disease and mortality. Findinghost proteins that can interact with virus proteins is helpful for the disease prevention andcan help us to futher understand the infection mechanism of the GCRV. The subject of thisexperiment is the grass carp reovirus strain096(GCRV096). The gene of VP6and NS38were cloned and expressed, the protein immunogenicity of the VP6and NS38was studiedas well in this study; This study has screened the host proteins that interact with GCRV096VP6and NS38proteins by yeast two-hybrid system.Depending on gene cloning of VP6and NS38, prokaryotic expression plasmidpET28a-VP6and pET28a-NS38were constructed, and then was respectively transferredinto E. coli BL21(DE3) for expression and purification of the target protein. Purifiedtarget proteins were incubated with CIK cells for12h,24h and48h, then let virus infectCIK cells for13h for fluorescence quantitative analysis. The experimental results showedthat the immune strength of VP6and NS38proteins become more and more strong withthe incubation time. Both VP6and NS38protein have immunogenicity. Theimmunogenicity of VP6protein is better. Infect CIK cells (incubated48h with protein)using GCRV096for4h,8h,13h. Difference of VP6and NS38gene expression level influorescence quantitative was detected in CIK. Results showed that the strongest time forthe immunogenicity of VP6protein is8h, the strongest time for the immunogenicity ofNS38protein is4h. This study use molecular cloning techniques to construct bait plasmid pGBKT7-VP6and pGBKT7-NS38, and then transfer them into yeast strain Y2Hgold for self-activationand toxicity test. The results showed that the bait plasmid was successfully constructed.The full-length cDNA library of the grass carp kidney cells (CIK) has been constructedthrough SMART technology. The capacity of library is2.4×106, and the library titer is6.44×107cfu/mL. The yeast Y2HGold containing the recombinant vector of VP6genewas mated with the yeast Y187pre-transformed with cDNA library plasmid of CIK. Referto MatchmarkerTMGold Yeast Two-Hybrid System User Manual, positive clones wereobtained through screening diploid yeast cells on high selective medium. Yeast plasmidsthat were extracted from positive clone were transferred into E. coli. Plasmid DNA thatwas purified from E. coli and the bait plasmid were cotransferred into the yeast strainY2Hgold to determine the interaction. The inserted cDNA in the positive clones weresequenced and analyzed by bioinformatic method. Four positive clones were obtained inthe experiment of screening proteins interacted with VP6. The results showed that twocDNA sequences were identified. The protein encoded by one of cDNA fragments hashighly homology with glyceraldehyde-3-phosphate dehydrogenase(GAPDH), but anotherhas not found typical homologous proteins; Three positive clones were obtained in theexperiment of screening proteins interacted with NS38. The results showed that two cDNAsequences were identified. The protein encoded by one of cDNA fragments has highlyhomology with40S ribosomal protein S16subunit, but another has not found typicalhomologous proteins.The immunogenicity of GCRV096VP6and NS38protein was studied, and itprovided a theoretical basis for the immunity and prevention of grass carp hemorrhage.And the host proteins interacted with VP6and NS38protein were screened by yeasttwo-hybrid system, which may lay the foundation for further study on the biologicalfunctions of VP6and NS38protein and is of importance in exploring the infectionmechanism of GCRV.