| Bovine TB is mainly caused by Mycobacterium bovis which pose an infectious risk to cattle and humans. After infected with Mycobacterium bovis, cattle can elicite a Th1 cellular immune response charactered by IFN-γsecretion. IFN-γcan activate the microbicidal activity of macrophages to eliminate the pathogen. But pathogens usually can not be eliminated and be restricted in a certain region, then the pathogen lies latent in the host. Studies of tuberculosis have suggested a shift in dominance from a Th1 towards Th2 immune response that is associated with suppressed cell mediated immune responses and increased humoral responses as the disease progresses. IL-4 is the major cytokine of Th2 immune response. Therefore IFN-γand IL-4 is important in TB research. In this study, the recombinant bacteria were constructed to express IFN-γand IL-4, the recombinant proteins were used to develop monoclonal antibody. All these results could be useful for studying the pathogenicity and the immune mechanism of Mycobacterium bovis, and for developing the method and technique in the detection of Mycobacterium bovis, and furthermore for studying bovine immunological biology immune response and immune regulation.1. Prokaryotic expression of bovine IFN-γand IL-4 genes in recombinant E.coliRecombinant bacteria BL21(pGEX-6p-1-BoIFN-γ) and BL21(DE3)(pET-BoIFN-γ) were constructed previously. The recombinant proteins, named rGST-BoIFN-γand rHis-BoIFN-γ, are expressed in soluble form. After induced by IPTG, the two recombinant proteins were purified, and their sizes were consistent with the prediction.The IL-4 gene without coding signal peptides was amplified from pSP73-BoIL-4 by PCR, then inserted into prokaryotic expression vector pGEX-6p-1 and pET-30a(+). The recombinant plasmids pGEX-6p-1-BoIL-4 and pET-BoIL-4 were transformed into DH5αfor sequencing. After sequencing confirmation, the two recombinant plasmids were transformed into expression bacteria BL21 and Bl21(DE3) respectively, and then recombinant bacteria BL21(pGEX-6p-1-BoIL-4) and BL21(DE3)(pET-BoIL-4) were developed. The recombinant proteins, named rGST-BoIL-4 and rHis-BoIL-4, are expressed in inclusion body form. After denaturation and renaturation of inclusion body, the two recombinant proteins were obtained, and their sizes were consistent with the prediction.2. Production and characterization of monoclonal antibodies against bovine IFN-γand bovine IL-4To prepare monoclonal antibodies against bovine IFN-γand bovine IL-4, purified protein rHis-BoIFN-γand rHis-BoIL-4 were respectively used as immunogen to immunize subcutaneously 7-week-old BALB/c mice. The immune dose was 100μg recombinant protein emulsified in complete Freund's adjuvant for the first immunization and in incomplete Freund's adjuvant for the second injections, the recombinant proteins without emulsified by adjuvant were immunized intraperitoneally for the third time, the interval of two immunizations is 2 weeks. Then an intravenous dose of purified protein was administrated. After 3 days, splenocytes from immunized mice were fused with Sp2/0-Ag-14 myeloma cells. Purified rGST-BoIFN-γand rGST-BoIL-4 were used as detecting antigen respectively, and the supernatant of hybridoma clones was screened by indirect ELISA. Thirteen hybridoma cell lines secreting McAbs against BoIFN-γ, named 1C12, 1F7, 1G5, 3E6, 4D5, 5E11, 5G4, 6F8, 6G6, 7E9, 8D3, 8F8, 9G11 were obtained. The immunoglobulin subclass of McAb 5G4 was IgG2b, the others were IgG1. The ascitic titers of these McAbs were 640000, 160000, 640000, 320000, 160000, 320000, 2560000, 2560000, 320000, 640000, 80000, 2560000, 10000 respectively. Seven hybridoma cell lines secreting McAbs against BoIL-4, named 2B8, 4A10, 5D6, 5D8, 7G10, 8B7, 10F8 were obtained. The immunoglobulin subclass were IgG1. The ascitic titers of these McAbs were 5000, 160000, 10000, 640000, 5000, 40000, 5000, 40000 respectively. In Dot-ELISA, all McAbs could only react with the immunogen and the detecting antigen. Western-blot analysis confirmed that all McAbs could only react with the corresponding recombinant proteins. The McAbs also reacted with the standard recombinant bovine IFN-γor IL-4 with biological activity. All these results suggested that the specific McAbs against bovine IFN-γand IL-4 were developed, which are very useful in both fundamental and practical studies.
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