The Interaction Between Grass Carp Fibulin-4 Protein With Grass Carp Reovious Proteins

The fibulins is a family of secreted glycoproteins,associating with these matrix structures is mediated by their abilities to interact with many extracellular matrix constituents,mainly including basement membranes and extracellular matrix of micro fiber,elastic structure.Fibulins are not only as a structural ECM component but also as a modulator for various cellular processes,such as cell growth,differentiation,angiogenesis,and tumor growth.The molecular mechanisms of fibulin function in those cellular processes need to be further explored.However,their roles in skeletal development and diseases are still unclear.Ctenopharyngodon idella is the main freshwater aquaculture species in China,which occupies the first place in the whole country.It is still prevalent in the middle and lower reaches of the Yangtze river,affecting the whole country,with annual loss of more than 30%,which seriously endangers the development of grass carp breeding industry.The study of the interaction between the virus proteins and host proteins may be helpful to elucidate the biological function of viral proteins and the pathogenesis of virus.Using previous research results,screening a Fibulin-4 extracellular matrix protein interacts with GCRV-JX01 VP7 and GCRV-JX02 W VP56,further finding that the expression level of Fibulin-4 is higher in grass carp muscle,brain,intestine,which was consistent with the clinical hemorrhagic disease of blood muscle,blood operculum and bowel.we tried to use molecular biology and cell biological methods to confirm the interaction between Fibulin-4 and two kinds of GCRV outer caspid proteins.This study mainly include the following three aspects:1 Baculovirus expression analysis of outer capsid VP56 gene encoded by type II reovirus of Grass CarpThe GCRV JX02 W was separated and saved by our laboratory,according to JX02 W strain S7 gene sequence,the primer for cloning recombinant plasmids pFastbacHTA-S7 was designed,total RNA,which was extracted from CIK infected with GCRV JX02 W,were used as the template for amplifying the gene S7 by RT-PCR.S7 were cloned into pFastbacHTA expression vector.The pFastBacHTA-VP56 were transformed into E.coli DH10 Bac strain competent cells,coated plates,select monoclonal colony PCR.The recombinant bacmid DNA that was analyzed by PCR.Then,the recombinant bacmid DNA was transfected into SF9 cells.Positive SF9 cells transfected with bacmid DNA stopped growth and lysed at 96 h post transfection.Western blot results showed that the His-tag antibody could specifically bind to His-VP56 fusion protein with the molecular weight about 62 kDa,which was soluble protein,laying a solid foundation for the study of its biological functions and the further research on the interaction between virus protein and grass carp host cell protein and new genetic engineering vaccine based on baculovirus expression vector of GCRV.2 The primary research on interaction between Fibulin-4 and GCRV-JX01 VP7 and GCRV-JX02 W VP56Recombinant plasmid pEGFP-Fibulin-4 and pDsRed1N1-VP7 and pDsRed1N1-VP56 were successfully constructed.pDsRed1N1-VP7,pDsRed1N1-VP56 and pEGFP-Fibulin-4 were transferred to GCO respectively.Subcellular localization showed that virus protein VP7,VP56 and host Fibulin-4 protein were distributed in cytoplasm,both of them overlapped with Fibulin-4.The polyclonal antibody of Fibulin-4 was prepared by constructing plasmid pGEX-4T-3-Fibulin-4.The purified recombinant proteins were used to generate polyclonal antisera specific for Fibulin-4 in BALb/c mice.Purified expression of VP56 protein and laboratory expression of VP7 protein.Using dot blot-overlay,the prokaryotic expression and purification of GST and GST-Fibulin-4 were immobilized on Poly vinylidene fluoride(PVDF),after His-VP7,His-VP56 protein incubation,Positive signals were developed with anti-His monoclonal antibody,the results showed with the hybrid from low to high protein concentration,protein signal from weak to strong.Both His-VP7 and His-VP56 can be adsorbed on GST-Fibulin-4 protein,but not GST protein.The pull down assay was using the Pierce? pull-down PolyHis Protein,Protein Interaction Kit,both of the elution can be detected with GFP-Fibulin-4 proteins,not GFP,further confirming the interactions between Fibulin-4 and VP7,as well as VP56.3The expression of Fibulin-4 in response to virus infection and the effect of Fibulin-4 overexpression on virus replication.To understand the expression of host Fibulin-4 during GCRV-JX01 infection,using qRT-PCR and Western blot to detect the transcription and translation levels of Fibulin-4 during GCRV-JX01 infection after 0 h,6 h,12 h,24 h and 36 h.The results showed that the mRNA level of Fibulin-4 was immediately suppressed upon GCRV challenge,with an observable sharp reduction period of 6-12 h p.i.resulting in minimum expression level at 12 h p.i.In consistence with its transcriptional expression pattern post viral challenge,translational expression levels of Fibulin-4 was significantly inhibited following the onset of virus infection until reaching the minimum level at 24 h.After G418 screening GCO cells transfected with pEGFP-Fibulin-4,the stable cell line of GFP-Fibulin-4 was established.The VP7 level in infected GCO cells overexpressing Fibulin-4 was significantly higher than that in control cells,GCRV replicated in Fibulin-4-overexpressing GCO cells with a significantly faster speed and reached to a maximal level that was significantly higher than in control cells at 36 h.