Construction Of Infectious Bursal Disease Virus’ Highly Productive Cell Lines And Analysis Of Antigenicity For VP3 Protein

Infectious bursal disease(infectious bursal disease,IBD)is an acute infectious disease caused by infectious bursal disease virus(infectious bursal disease virus,IBDV),mainly infect 3-6 weeks chicks and multiply in the bursa.It could not only kill the infected animals,but also lead to immunosuppression and increase susceptibility to other diseases.Besides IBDV varied quickly and was difficult to eradicate after effected,so IBD is one of the most important infectious diseases bringing major harizonaards to poultry.The detection of IBDV antibody and vaccine of flocks are effective methods to prevention widespread of IBD.In this study,we conducted experiments into two parts for the control of IBD.One hand,the DF-EGFP-miR-9 cells overexpressing gga-miR-9 was built through lentiviral expression system and was purified by FCM.The virus title of cell culture was detected to estimate the possibility of DF-EGFP-miR-9 cells for virus production.On the other hand,the VP3 protein was obtained through eukaryotic expression system,and VP3-ELISA was established to detect the level of IBDV antibody.Part Ⅰ:Construction and purification of DF-1 cells overexpressing gga-miR-9 geneIn order to explore the ability of DF-1 cells overexpressing gga-miR-9 gene in effecting the replication of infectious bursal disease virus(IBDV),the pri-gga-miR-9 gene was amplified from the chicken genome,and the recombinant lentiviral vector plasmid pL-EGFP-miR-9 was successfully achieved in the experiments.The recombinant lentiviral VL-EGFP-miR-9 was generated from 293FT cells co-transfected with pC-GP,pR-Rev,pVSVG and pL-EGFP-miR-9,then the DF-EGFP-miR-9 cells were gained from DF-1 cell line infected with VL-EGFP-miR-9 by blasticidin selection and flow cytometry(FCM).The sorted DF-EGFP-miR-9 cells with normal morphology has greater fluorescence intensity and density than the unsorted DF-EGFP-miR-9 and normal DF-1 cells under the fluorescence microscope,and all of them have a similar growth curve.The real time RT-PCR assay indicated that the expression of gga-miR-9 of the sorted DF-EGFP-miR-9 cells are 500 times higher than normal DF-1 cells.Furthermore,the sorted DF-EGFP-miR-9 cells has higher virus titer(109.25 TCID50/0.1mL)than unsorted DF-EGFP-miR-9 cells(108 TCID50/0.1mL)and normal DF-1 cells(106.875 TCID50o/0.1mL).These finding suggest that the sorted DF-EGFP-miR-9 cells can stably overexpress gga-miR-9 and significantly improve the virus titers of cell culture.Part Ⅱ:Expression of infectious bursal disease virus vp3 gene in insect cells and the antigenicity for one indirect-ELISAIn order to analyze the antigenicity of infectious bursal disease virus(IBDV)recombinant VP3 protein for establishment of ELISA,one recombinant vBac-vp3 baculovirus was successfully achieved in the experiments.The IBDV vp3 gene was inserted into the baculovirus expression system donor pFastBacHTA plasmid.The recombinant baculovirus expression plasmid pBac-vp3 was obtained after the recombinant donor plasmid was transformed into competent pFastBacHTA-vp3 E.coli DH10Bac bacteria and cultured in selective media.One recombinant vBac-vp3 baculovirus was obtained by the pBac-vp3 plasmids were transfected into Sf9 insect cells and cultured.The expressing recombinant VP3 protein(about approximate 33ku)was positively detected by Western blot,and specific fluorescence was also detected out by the indirect immunofluorescence assay(IFA)in Sf9 cells which infected with vBac-vp3.An indirect-ELISA based on the expressed VP3 protein as the coating-antigen was set up for the detection of the IBDV antibodies compared to the VP2-ELISA coated with recombinant VP2 protein and IDEXX-ELISA coated with whole virus.The results showed that the positive detection rate and the ability to distinguish between positive and negative sera of VP3-ELISA is lower than the VP2-ELISA and IDEXX-ELISA;the correlation coefficient of VP3-ELISA and IDEXX-ELISA is lower than VP2-ELISA and IDEXX-ELISA’s in the IBDV antibodies detection of 42 chicken sera.This study indicated that the recombinant VP3 protein expressed in insect cells possessed specific antigenicity and can be used for detecting IBDV antibodies in ELISA,but is weaker than the VP2 protein and the whole IBDV virus.