Clone Of Goose Immunoglobulin Light Chain With λ Type And Application Of Specific Antibody Prepared Against Its Constant Region

Immunoglobulin (Ig) is an important effector molecule of humoral immunity, which plays an important role in immune regulation. There has no report of the goose immunoglobulin so far at molecule level.First, the primers for PCR amplication were designed according to the cDNA sequences of chicken Ig light chain (ChIgL) and duck Ig light chain (DuIgL) in GenBank, the goose Ig light chain (GoIgL) cDNA sequences was first cloned by RT-PCR and Nested PCR combinated in the world. Then the character of sequence cloned here was analyzed, such as the comparison of the sequence, similarity of amino acid sequence, prediction of signal peptide, glycosylation sites,3D struction, construction of phylogenetic tree, which indicates the sequence cloned here was belong the λ type of GoIgL.Second, a pairs of expression primers specific for GoIgCL were designed according to the cDNA sequence cloned here, then the recombinant protein rGoCL was expressed in E. coli RosettaTM(DE3)plysS and the PAb against GoIgCL was prepared with rGoCL. The antigen-antibody reaction with avian IgL was analyzed using the PAb against GoIgCL, which further confirmed the presence of common B-cell epitope in avian IgCL.At last, the BALB/c mice were immuned with goose serum Ig purfied by Protein A affinity chromatography system, two hybridoma cell lines (1B11and3G9) were selected to secrete antibody against GoIgCL, then the biological characterization was identified. The results of indirect immunofluorescence, Western blot and indirect ELISA showed that the1B11and3G9hybridoma cell lines secret antibody against GoIgCL with high specificity, having no reaction with the serum of other species. The monoclonal antibody (MAb) against GoIgCL was used as the ligands to prepare affinity chromatography, which can purify the goose Ig from serum; The HRP-labeled MAb against GoIgCL can detect the fluctuation of specific antibodies in goose body after immuned BSA and GPV VLPs, seperately; An ELISA method for quantitative determination of the content of goose Ig with MAb against GoIgCL was established, which was used to detect the goose Ig from serum.In summary, we cloned the gene of GoIgL for the first time and the character of GoIgL was analyzed by bioinformatics software. Then the specific antibody against GoIgCL was prepared with recombinant protein expressed containing GoIgCL cloned here, subsequently the antibody was used for immunoassay, which laid the foundation for the research of goose basic immunology.