Cloning And Expression Of Bovine Interferon-Gamma CDNA And Development Of Monoclonal Antibodies Against The Expressed Product

Interferon-gamma, characteristically produced by Th1 cells, CD8+ cells and natural killer cells, is a pleiotropic cytokine and plays a critical role in anti-virus, anti-tumor and immune regulation. It was shown that the level of endogenous IFN- γ could reflect the status of cellular immunity of one body to some extent, while the exogenous EFN- γ could be used as therapeutic drug in the treatment of infectious diseases or tumors, or as a vaccine adjuvant to promote vaccination efficacy. Consequently, the McAb specifically against IFN- γ is a very important tool in the field of EFN- γ research, such as study of immunomechanism, detection of immune status, and purification of rIFN- γ. DFN- γ and its McAbs are very useful in veterinary field too. However, it is rather difficult to obtain enough BovIFN- γ for clinical use or to prepare McAbs by the traditionally time-consuming methods. In recent years, with the advancement of the molecular biolology, it is feasible to obtain rBovIFN- γ through genetic engineering and by which to prepare its McAbs. This study was aimed to develop McAbs against BovIFN- γ through using prokaryotic expression rBovIFN- γ and recombinant eukaryotic expression plasmid, which will facilitate the further research on application of rBovIFN- γ and its McAbs in diagnosis, prevention and control, treatment and other associated study of bovine diseases.1. Expression and identification of recombinant bovine interferon-gamma in E. coliFull length cDNA of bovine interferon-gamma precursor (PreBov-IFN- γ) was amplified by reverse transcription-PCR from total RNA of bovine spleen lymphocytes stimulated with Concanavalin A (Con A), and cloned into pVAXl plasmid. The DNA fragment of mature BovIFN- γ (without signal peptide) was amplified by PCR from recombinant pVAX-PreBovEFN- γ , and then subcloned into pET-30a(+) andpGEX-6P-l respectively. After confirmation by sequencing, the two recombinant plasmids, pET-30a(+)-BovIFN- γ and pGEX-6P-l-BovIFFN- γ, was transformed into Kcoli BL21(DE3) or E.coli BL21. When recombinant bacteria was induced by EPTG, the anticipated MW 22 kD or 42 kD protein band appeared on SDS-PAGE gel, which accounting for up to about 60% and 45% of total bacterial proteins under optimized conditions. The purified fusion protein, rHIS-BovIFN- γ and rGST-BovIFN- γ , showed antiviral activity in the vesicular stomatitis cytopathic effect reduction assay and the titers were up to 1.97 X10⁵ U · mg-1 and 3.2 X104U · mg-1 respectively.2. Development and identification of monoclonal antibodies against recombinant bovine interferon-gammaTo prepare monoclonal antibodies against rBovEFN- γ, 6-week-old BALB/ c mice were immunized with recombinant plasmid pVAXl -PreBovIFN- γ and purified fusion protein rHis-BovIFN- γ respectively. Splenocytes from immunized mice were fused with Sp2/0-Ag-14 myeloma cells, the supematants of hybridoma clones were screened by indirect ELISA. Four hybridoma cell lines were obtained and named 4C7, 3H5,2D9, 3E4. The isotypes of 4 McAbs were IgM, IgG₂b, IgG₁, IgM, and the ELISA titers of the 4 McAbs' culture supernatant were 1:1600,1:3200,1:1600,1:1600, and those of the ascites were 1:25600,1:51200,1:25600,1:12800 respectively. Western-blotting analysis confirmed that the 4 McAbs could only react with the 22kD band of rHIS-BovIFN- γ and 42kD band of rGST-BovEFN- γ. In Dot-ELIS A test, the 4 McAbs could only react with purified rHIS-BovIFN- γ and rGST-BovIFN- γ but not with rHIS-ChlFN- γ, rGST-CMFN- γ and rHIS-ChIFNIL-2. All these results showed that 4 hybridoma cell lines secreting high-titer and high-specific monoclonal antibodies against rBovIFN- γ have been successfully developed, which provides a basis for the further research and clinical applications of the McAb against BovIFN- γ, such as rBovIFN- γ purification and diagnosis of bovine diseases.