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

Interferon-gamma (IFN-γ), produced mainly by Th₁ cells, CD8⁺ T cells and natural killer cells, plays a critical role in anti-virus, anti-tumor, and immune modulation. Its immune modulation includes activating macrophages, upregulating the expression of MHC class I and class II molecules, prompting the presentation of antigens, and inducing Th cell growth and differentiation. The level of IFN-γ is an important index to the cellular immunity. Consequently, monoclonal antibodies (McAbs) specifically against IFN-y are very valuable in detecting and evaluating the cellular immune status. In this study, recombinant plasmids of chicken IFN-y (ChlFN-γ) were constituted and expressed, and hybridoma cell lines secreting McAbs against ChlFN-γ were generated. The results provide helpful information for constituting the methods and techniques used in the detection and analysis of immune cells and immune regulation by utilizing these McAbs in chicken model. 1. Cloning and expression of chicken interferon-gammaSpecific primers were designed and synthesized according to the previously published sequence in GenBank and used to amplify cDNA fragments of ChlFN-y precursor (PreChIFN-γ) and mature ChIFN-γ (without signal peptide sequence). Full length cDNA of PreChIFN-γ, amplified by reverse transcription-polymerase chain reaction (RT-PCR) from total RNA of chicken splenocytes stimulated with Concanavalin A (Con A), was cloned into pGEX-6P-l and the recombinant plasmid pGEX 6P-l-PreChIFN-γ was transformed into E.coli BL21. PreChlFN-y gene was identified by restriction enzyme digestion and sequencing. The recombinant bacteria was named asBL21 (pGEX 6P-1 -Pre-ChlFN-γ).cDNA fragment of mature ChlFN-γ was amplified by PCR from plasmid pGEX 6P-l-PreChIFN-γ, then subcloned into pGEX 6P-1 and pET-30a(+), respectively. Both of the recombinant plasmids DNA were identified with restriction enzyme digestion. pGEX 6P-l-ChIFN-γ and pET-ChlFN-γ were then transformed into E.coli BL21 and BL(DE3), respectively, constituting recombinant bacteria BL21(pGEX 6P-l-ChIFN-γ) and BL21(DE3)( pET-ChlFN-γ).After induced by IPTG, the three recombinant bacteria, BL21(pGEX 6P-l-PreChIFN-γ), BL21(pGEX 6P-l-ChIFN-γ) and BL21(DE3)( pET-ChlFN-γ) were analyzed by SDS-PAGE, showing that fusion protein GST-ChlFN-γ, His-ChlFN-γ were expressed as 42kD and 24kD, respectively. After they were disrupted by sonication, SDS-PAGE experiments indicated that fusion protein GST-ChlFN-γ expressed in BL21(pGEX 6P-l-PreChIFN-γ) can only exist as inclusion body, and the fusion proteins of BL21(pGEX 6P-l-ChIFN-γ) and BL(DE3)(pET-ChIFN-γ) existed as both soluble protein and inclusion body.In the vesicular stomatitis virus cytopathic effect reduction assay, purified GST-ChlFN-γ had antiviral activity and the titer was approximately 1.2 x 10⁴U/mg. 2. Development and characterization of monoclonal antibodies against chicken interferon-gammaTo prepare monoclonal antibodies against ChlFN-γ, the inclusion body of the recombined bacteria, BL21(DE3)(pET-ChIFN-γ), was used as immunogen to immunize intraperitoneally 8-week-old BALB/c mice. The immune dose was 300μg of the ChlFN-γ inclusion body, emulsified in complete Freund's adjuvant for the first immunization and in incomplete Freund's adjuvant for the next three injections with 2-week intervals. Then an intravenous dose of the inclusion body was administrated. After 3 days, splenocytes from immunized mice were fused with Sp2/0-Ag-14 myeloma cells. Purified GST-ChlFN-γ was used as a detecting antigen, and the supernatant of hybridoma clones were screened by indirect ELISA. The specificity of the McAb was then characterized by indirect ELISA, Dot-ELISA and Western-blot. Two hybridoma cell lines secreting McAbs against ChlFN-γ named 1G5, 5E3 were obtained. The immunoglobulin subclasses of 2 McAbs were IgG2a, and the ELISA titers of these McAbs ascites were 1.6x10⁶, 1.2 x 10⁵ respectively. In Dot-ELISA, the 2 McAbs couldonly react with BL21(DE3)(pET-ChIFN-γ), BL21(pGEX 6P-l-ChIFN-γ), which expressed His...