Screening And Preliminary Identification Of Cell Receptors Of Grass Carp Reovirus

Grass carp(Ctenopharyngodon idellus)is China’s important freshwater aquaculture species,widely distributed in north and south waters in China,which is one of four major Chinese carps.Grass carp hemorrhagic disease caused by Grass Carp Reovirus(GCRV)has brought huge economic losses to Grass Carp aquaculture in China.There is no effective prevention and treatment to deal with the raging GCRV.Good disease resistance of fish cultivation and targeted therapy are two important ways of prevention and treatment of grass carp hemorrhagic disease.Therefore,GCRV and CIK cells were as the research object in this study,through the study of target protein,exploring virus invasion mechanism,to provide better prevention and control of grass carp hemorrhagic disease,also provide a theoretical foundation for anti-GCRV grass carp breeding.In this study,the preparation of polyclonal antibody against GCRV virus particles,using two-dimensional electrophoresis and virus overlay protein binding assay to screen the virus receptors,receptor molecules were identified by mass spectrometry.Finally we analyzed the chose molecular receptor,in order to verify that the role of the receptor molecule in GCRV invasion CIK.The results obtained in this work were as follows:1.GCRV was purified by nonlinear sucrose density gradient centrifugation,and experimental rabbits were immunized with GCRV virus particles by back multi-point injection,in order to prepare polyclonal antibody against GCRV.The titer of polyclonal antibody reached 1:102400 by ELISA method,using western blot experiment to analyze the specificity of the antibody,and the polyclonal antibody could react five belts of protein with GCRV virus particles.2.Utilizing virus overlay protein binding assay to screen potential receptor molecules of GCRV in CIK cells,got a specificity protein band about 39 kDa.A specific protein dot was displayed by two-dimensional electrophoresis and virus overlay protein binding assay.This protein dot was identified by mass spectrometry and 12 proteins could be matched.For the identified 12 proteins,comprehensive consideration of protein synthesis score,biological functions and the research status,this article chose HSP70 protein for further study.3.CIK cells were analyzed by immunofluorescence,flow cytometry and western blot assays.The results revealed that HSP70 protein existed in the cell surface of CIK cells.The toxicity of HSP70 protein inhibitor(VER155008 and pifithrin-μ)in CIK cells was determined by MTT method,and it would not affect the cell vitality when each drug concentration was lower than 15 μM.GCRV infected CIK cells which were incubated with HSP70 protein inhibitors and GCRV VP6 gene expression was detected by RT-qPCR.The results showed that with the increase of drug concentrations,VP6 gene expression quantity decreases,and when VER155008 and pifithrin-μ(1:1)were used at the concentration of 15 μM,VP6 gene expression down nearly 90%.In summary,HSP70 plays an important role in GCRV invasion CIK cells and it is one of the receptor for GCRV infection CIK cells.This study laies a foundation for the mechanism study of CIK cells during GCRV infections.