Construction Of A Cell Line Stably Expressing The VP2 Protein Of Avian Infectious Bursal Disease Virus And The Isolation And Identification Of Suspected IBDV Receptors

Infectious bursal disease is a highly contagious disease of poultry,causing severe immunosuppression and bursal injury,which was found at the town known as Gumboro in the United States in the earliest 1957,therefore it is called Gumboro disease.Infectious bursal disease virus belongs to the Birnavirus family containing two serotypes,but only serotype one has pathogenicity to the chickens.The virus has non-enveloped and only one layer of shell.it express 5 proteins,named VP 1,VP2,VP3,VP4 and VP5;VP2 is the main host protective antigen and the composition of the outer shell.Infectious bursal disease virus is a member of double stranded RNA virus,just like many members as the Reoviridae family,they all have a certain similarity.A lot of double stranded RNA virus receptors have been identified;for example,rotavirus receptors including sialic acid,integrin family,heat shock cognate protein 70,Toll-like receptor and tissue blood type antigens,etc.,they interact with different proteins of this virus,thus infecting the different tissues and cells,for example,reovirus uses the junctional adhesion molecule A and the Nogo as the receptors,and then GM2 polysaccharide and sialic acid are used as co-receptors to assist the effect of virus on host cells causing virus infection and replication.Infectious bursal disease virus has been studied by many scholars.Now many isolated receptors have been identified,the four kinds of receptors such as surface immunoglobulin M,integrin,annexin A2,heat shock protein 90 have been found from different cells;the surface immunoglobulin M from chicken B cell lines has found in LSCC-BK3,chickens Hsp90 is found at DF-1 cells,integrins are verified by BALB/c to 3T3 cells,annexin A2 of chicken is captured by use of the VOPBA experiment in DF-1 cells.For many decades from the finding of the IBDV,the variations from avirulent to virulent strains,from wild strain to cell adapted strain and so on lead to different strains have the same pathogenicity to the same cell or different infectivity to the different cellse.This maybe have some relationships with the receptor diversity.1.Construction of DF-1 cell lines stably expressing VP2 protein of infectious bursal disease virusThe eukaryotic expression plasmid of pcDNA-VP2 was first constructed in this study,and then this vector was transfected into DF-1 cells by liposome transfection.VP2 gene of infectious bursal disease virus in this vector was inserted into the chromosomes of the DF-1 cells by means of homologous recombination in the process of cell division.And the drug of zeocin was used to screen the clone of DF-1 cells for stable expressing VP2 proteins.Through a series of research work,the final concentration of 25?g/ml zeocin was determined for the best screening concentration.When the cell lines were subcultured to the 60 generations,the constructed cell lines were analyzed and identified by use of the kinds of methods such as the cell morphology,the cell growth curve,the indirect immunofluorescence,the polymerase chain reaction(PCR)and the western-blotting methods.It was found that the the cells had a good growth state and the morphology was just like DF-1 cell line because the constructed cell line had the original spindle characteristics except slightly hypertrophy.On the resistant drug-maintained condition,by detecting and comparing the growth curve,the rate of the cell growth and the differentiation were lower than that of the normal DF-1 cells and the non resistant drug-maintained cells.And the VP2 gene was found and detected by PCR in the genome from the extraction of the cells.The fully expression VP2 proteins of infectious bursal disease virus were found by using monoclonal antibody 2H11,indirect immunofluorescence assay in pcDNA-VP2 DF-1 cell line,and Western-blot experiments.These series of experiments showed that the pcDNA-VP2 DF-1 cell line was constructed successfully and the target protein VP2 was stably expressed.This cell line provided an important experimental material and laid a foundation for the next experiment.2.Isolation and identification of infectious bursal disease virus receptor on DF-1 cellsThe immunoprecipitation was performed by the extraction cell membrane proteins from pcDNA-VP2 DF-1 cells and DF-1 cells(control)f interacted with the purification of monoclonal antibody 2H11 and the protein A+G Agrose proteins,then Precipitate product was analyzed by SDS-PAGE electrophoresis and the differential protein bands were identified by silver staining.In addition,the cells of the pcDNA-VP2 DF-1 and the DF-1(control)were fully lysed and the protein in the lysis supernatant also was interacted with the above 2H11 and protein A+Gagrose.Then co-immunoprecipitation was performed,SDS-PAGE electrophoresis and silver staining also were used to analyze co-immunoprecipitation protein.By comparing the differentially expressed proteins,we excised the different proteins.The proteins from the isolation of the membrane immunoprecipitation were combined with that from the lysis supernatant immunoprecipitation.The mixture was used to be analyzed by mass spectrometry to yield nearly 50 kinds of proteins,and then these 50 proteins were classified and the function of each protein was analyzed,especially in if it had the ability to act as a receptor involved in the virus infection and replication.We identified HSC70 and vimentin as putative receptors by comparing the final screen.This established foundation for the further identification and verification of the receptors.3.Verification and correlation analysis of HSC70 and vimentin receptors(1)HSC70 antibody was used to block DF-1 cells,and then the virus blocking experiment was carried out to investigate the result of blocking,especially in the effect on the infection and the replication of the infectious bursal disease virus.The process is as follows:the DF-1 cells were incubated with HSC70 antibody in advance,and the HSC70 receptor was blocked by antibody neutralization,and then cells were infected with the cell-adapted virus.After 48 hours,the pathological changes of the cells was observed and indirect immunofluorescence assay,fluorescence quantitative PCR test,Western-blot test,TCID50 were performed.Compared with that of the positive control,it was found that with the increase of antibody concentration,the inhibition effect on virus was enhanced,The main result of the test as follows:the extent of cytopathic effect was decreased,the expression levels of the viral gene and the protein were declined,the titer of virus was reduced obviously.(2)DF-1 cells were treated by the HSC70 inhibitor of VER-155008,and then the virus infection experiment was performed.The aim for this experiment was mainly to observe that after HSC70 was inhibited by the heat shock protein 70 inhibitor of VER-155008,it effected on the infection and the replication of IBDV.This experimental steps were as follows:firstly,DF-1 cells were incubated with inhibitors for 6 hours,then added IBDV cell-adapted virus to be infected.At 24 h,48 h,and 72 h of the different time points,the TCID50 in the supernatants was measured and the cell lysis was analyzed by Western blotting.Compared with that of the positive control,it was found that HSC70 of the cells in the experimental group after HSC70 inhibitor treated was inhibited greatly at different times,and that the virus titer and the expression of viral proteins also were significantly decreased(3)the cells of DF-1 was inhibited by the inhibitor of vimentin,and then infected with infectious bursal disease virus.This experiment is as follows:DF-1 cells were pretreated with acrylamide,an inhibitor of vimentin,for 2 hours,and then added IBDV cell-adapted virus to be infected.At 24 h,48 h,and 72 h of the different time points,the virus titer in the supernatant was measured and the cell lysis was analyzed by Western blotting.Compared with that of the positive control group,a significant decrease of the viral replication ability and of the expression of viral protein in the cells were found in the experimental group.4.HSC70 and vimentin receptors reconstitution experimentsHSC70 and vimentin genes were amplified by RT-PCR,and then were cloned into the eukaryotic expression vector of pcDNA3.1 respectively to construct the eukaryotic expression plasmids of pcDNA-HSC70 and pcDNA-vimentin.MDCK cells,non-susceptible to IBDV,were transfected with these constructed vectors.After 48 hours later,the transfected cells were infected with IBDV(MOI=5).After 7 days later,the TCID50 was tested in the supernatant and cell lysis in the cells,the indirect immunofluorescence was performed for the normally infected cells Compared with that of the normal DF-1 cells,the results all showed that TCID50 was 0,indirect immunofluorescence was negative.From these results,it seems to appear that receptor reconstruction failed,but after consulting the relevant literature,it was found that the expression of HSC70 in vitro can produce natural immunity,mediating type I interferon secreteell having antiviral effect.Moreover,and the vimentin can form a "cage like structure",and the over expression of the protein can isolate the virus and prevent it from entering the cells.