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Establishment of Detection for Bovine IFN-γ with the Recombinant Specific Receptor

Author: FanLei
Tutor: BuZhiGao
School: Chinese Academy of Agricultural Sciences
Course: Preventive Veterinary Medicine
Keywords: Bovine IFN-γ Acceptor Reorganization Double sandwich ELISA
CLC: S852.4
Type: Master's thesis
Year: 2008
Downloads: 60
Quote: 1
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Abstract


Is in the role of a specific inducer of interferon (interferon, IFN), produced by cells having a high degree of biological activity of the glycoprotein, having immune regulation and anti-viral activity in the cells of the same animal species. The level of endogenous IFN-γ levels largely reflect the immune status of the body's cells. IFN-γ quantitative analysis techniques, diagnostic immunology basic research, as well as Mycobacterium tuberculosis and brucellosis and other intracellular pathogenic infection with a highly specific, sensitive to highlight the advantages of broad application prospects. Bovine IFN-γ receptor extracellular region fragment containing the signal peptide of the eukaryotic expression plasmid is first constructed in this experiment 293T cells transiently transfected mammalian cells, the recombinant baculovirus expression of bovine IFN-γ as a ligand, the package the affinity and the ability to detect by the ELISA plates, the results show, mammalian cells, recombinant expression bovine IFN-γ receptor fragment expression product can be secreted into the culture supernatant, and having a good affinity with bovine IFN-γ and response capabilities. Further constructed prokaryotic expression plasmid expressing bovine IFN-γ receptor extracellular region fragment and transmembrane - intracellular region fragment and transformed into E. coli BL21 induced by IPTG, SDS-PAGE and Western-blotting confirmed the target protein to be successful expressed. The expression of the receptor protein is obtained through a Glutathione Sepharose 4B affinity chromatography purification. On this basis, the use of the expression of the purified recombinant bovine IFN-γ receptor extracellular region fragment and transmembrane - intracellular region fragment instead of the traditional double sandwich ELISA technique coated antibody (mAb), bovine IFN- double sandwich ELISA quantitative detection method of gamma interferon on the reaction conditions were optimized. Recombinant receptor to react with a variety of other cytokines were negative, showing good specificity. Using the method of detection of prokaryotic expression system of bovine IFN-γ receptor extracellular domain of recombinant proteins and transmembrane - District Reorganization intracellular protein quantitative standard curve linear regression coefficients were 0.9683 and 0.9722, sensitivity can reach 40.65 ng / mL; for detecting the recombinant baculovirus expression of bovine IFN-gamma receptor extracellular outer zone intracellular region of the recombinant protein and the transmembrane - recombinant protein quantitation standard curve of the linear regression coefficient of 0.9656 and 0.9820, respectively, detection sensitivity sex up to 10 anti-viral activity units (IU) / 0.1ml. As controls, U.S. ADL production commercialization bovine IFN-γELISA detection kit to detect recombinant baculovirus expression of bovine IFN-gamma, the sensitivity was 80 IU/0.1ml. The results of this study indicate that the recombinant expression of bovine IFN-γ receptor having a specific affinity for the package is alternative to the traditional double sandwich ELISA technique antibody (mAb), bovine IFN-γ in a double sandwich ELISA established quantitative detection method has good sensitivity and specificity of the method, with the the traditional antibody (mAb) package double sandwich ELISA, compared with the preparation of simple, low-cost advantage. The establishment of the IFN-γ interferon receptor affinity principle detection method for the detection of other cytokines technology has opened up new ideas.

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CLC: > Agricultural Sciences > Livestock, animal medicine,hunting,silkworm,bee > Animal Medicine ( Veterinary Medicine) > Basic Veterinary Science > Animal Immunology
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