ELISA Techniques Based On Recombinant Protein Of IBV And Gene Immunization Of IBV

Several IBV isolates causing severe nephritis in chickens were isolated from diseased chicken flocks in China. With specific primers designed according to the genome of published IBV strains, the S1 gene and N gene of the IBV isolates were cloned and sequenced. Analysis of the gene sequence with DNAstar software showed that the IBV strains with kidney tropsim isolated in China recently shared a high identity(82.9%-98.6%) while they shared low identity with traditional IBV strains. A lot of point mutations were found in the S1 gene and N gene of the IBV isolates. In addition, a insertion mutation of 21-nt were found in the S1 gene of two IBV isolates, SC021202 and J. These results indicated that the IBV strains SC021202 and J with kidney tropsim epidemic in China currently were variant strains.The N gene of IBV were expressed in E coli. with pBAD/his sysytem. The yield of the expressed N protein to the total bacterial proteins was about 20%. In addition, S1D segment and S1F segement of S1 protein of IBV were expressed in Ecoli. with pGEX expression system. The S1D protein and S1F protein contains the D epitope and F epitope of S protein of IBV respectively. Both of the N protein and S1 protein expressed in vitro was able to react specificly with anti-sera against IBV, which suggested that they mocked the antigenicity of native protein to some extent. With a Ni+ column, N protein, S1D protein and S1F protein were successfully purified. What's more, S1 glycoprotein was also expressed in yeast strain X33 with ppPICZ α B vector which was transformed into X33 by electroporation..With the purified N protein as coating antigen, an indirect ELISA assays(N-ELISA) were established which could detect the antibody in chicken serum against N protein of IBV. The reaction conditions of N-ELISA were optimized. Dilution of N protein to a final concentration of 0.19μg per well with PH8.5 Tris-HCl was found to be the best coating condition. The primary antibody(serum sample) and secondary antibody(HRP-labeled rabbit anti-chicken serum) were diluted to 1:100 and 1:800 respectively in N-ELISA. Detection of antisera against IBV and against other pathogens showed that N-ELISA was specific for IBV. In an experiment of clinical samples detection, N-ELISA shared 95.7% identity of total positive ratio with a commercial ELISA kit+(IDEXX). In addition, the S1F protein expressed in Ecoli was used as coating antigen, an indirect ELISA(S1F-ELISA) was established which could detect specific antibody against S protein of IBV. As described above, the reaction of S1F-ELISA was optimized too. The antigen was diluted to 0.6μg per well with PH8.5 Tris-HCl for coating. Dilution of primary antibody and secondary antibody were diluted to 1:50 and 1:800 respectively. Detection of antisera against IBV and against other pathogens showed that N-ELISA was specific for IBV. In an experiment of clinical sample detection, S1F-ELISA shared 100% identity of total positive ratiowithlDEXXELISAkit.Purified N protein, SID protein and S1F protein were injected into BALB/c mouse as antigen. Then 17 hybridoma cell lines in total secreting monoclonal antibody(Mab) were prepared by hybridoma technique and all of the Mab were IgGl,K subtype. Among the 17 Mabs, eight clones were against N protein, three clones were against SID protein and the other six clones were against S1F protein. The specificity of all of the Mabs was confirmed by western-blotting and ELISA. The ELISA titer of the 17 Mabs varied from 103 to 106. In addition, polyclonal antisera against N protein, SID protein and S1F protein were also prepared respectively by vaccination of SPF chickens with purified proteins and the antibody was titered by agar gel precipitation(AGP) assay.SI gene and N gene were ligated into mammalian expression vector pCI-neo respectively. The recombinant plasmids were transfected into Vero cells and the expression of SI protein and N protein was confirmed by indirect immunofluorescentce technique(IFA) and immunocytochemistry(ICC). The recombinant plasmids were used as DNA vaccine and their immune efficiency were evaluated in SPF chickens. The results indicated that the DNA vaccine against IBV based on SI gene and N gene could provide protection for chickens against virulent IBV challenging. Lymphocyte prolification assay and neutralization assay showed that SI gene could stimulate both cellular immunity and humoral immunity. SI gene induced VN antibody while N gene couldn't. It was possible that N gene provided protection via celluar immunity. Chicken IL2 was used as adjuvant in DNA vaccine experiment. Plasmids containing IL2 gene or recombinant IL2 protein could both improve efficiency of DNA vaccine against IBV. The plasmids worked better than IL2 protein did. In addition, combination of IL2 plasmids and SI plasmids worked better than co-expression of IL2 gene and SI gene in a single plasmid.