Establishment Of Detection For Bovine IFN-γ With The Recombinant Specific Receptor

Interferon (IFN), generated by specific cells induced by inducer, is a kind of glycoprotein that possesses high bioactivity and broad-spectrum antiviral activity on the homogeneity animal cells. The quantitative determination of endogenous IFN-γconcentrations has very significant value in immunological basis; has high specific,senceitive advantage and wide prospects on intracellular pathogen diaglosis such as mycobacterium tuberculosis and brucella.We constructed Eukaryotic expression plasmid includes the extracelluler region and signal peptide. It was transfected into 293T cells to carry out transient expression. The cell supernatant was coated to the ELISA plate and tested the affinity to BoIFN-γby ELISA. The results were confirmed that the interesting protein was expressed in 293T cells and the mature protein was secreted out of cells by signal peptides and the affinity to BoIFN-γis very good. We constructed prokaryotic expression plasmids were constructed, respectively, the plasmids include the extracelluler region without signal peptide and the transmembrane domain and intracellular region, then transformed into host bacteria BL21 that were then induced by IPTG. SDS-PAGE and Western-blotting were carried to assure that the interesting proteins were expressed. The proteins were purified by Glutathione Sepharose 4B. And on the basis, we use the affinity between IFN and receptors instead of the specificity binding of antigen and antibody, and establish a capture enzymelinked immuno sorbent assay (ELISA) for detection of bovine IFN-γand optimized the conditions. The recombinate protein didn't reaction with other cytokines, that is, they have high specific advantage. We used the quantitated bovine IFN-γwhich was expressed from bacteria BL21 as the standard preparation, and draw the standard curve of quantity. The linear regression coefficient of the recombinate protein of extracelluar region and the transmembrance domain and intrancelluar region were 0.9683 and 0.9722. The sensitivity value of bovine IFN-γwas 40.65ng/mL. The antiviral activities of bovine IFN-γwhich was expressed from recombinant baculovirus in sf9 cells was measured, and the bovine IFN-γwas used as the standard preparation, and draw the standard curve of antiviral activities. The linear regression coefficient of the recombinate protein of extracelluar region and the transmembrance domain and intrancelluar region were 0.9656 and 0.9820. The sensitivity value of bovine IFN-γwas 10IU/0.1mL. As control, we detected the bovine IFN-γwhich was expressed from recombinant baculovirus in sf9 cells with the commercial ELISA kit of bovine IFN-γof Amercian ADL, the result showed that the sensitivity value of bovine IFN-γwas 80IU/0.1 mL.Our study shows that the recombinate protein has specific,high affinity advantage. We established the quantitative capture ELISA which uses the recombinate protein as capture antibody instead of the conventional capture antibody. Compare with the conventional capture ELISA, our detection has the ascendancy of convenient and low price. Moreover, the detection which based on the affinity of the receptor gives a new way to detect other cytokines.