Expression and Activity Determination of Bovine Interferon-gamma Gene

With the reverse transcription polymerase chain reaction (RT-PCR), the DNA sequence encoding the cattle’s interferon-gamma (BovIFN-γ) and the signal peptide was amplified, from the total RNA of the lymphocytes stimulated with concanavalin A in the peripheral blood of bovine, which was then cloned into vector pMD18-T and sequenced. The sequencing result showed that there is 100% homology among the documented sequences and sequence reported here, which was successfully inserted into the expressing plasmid pRLC and was highly expressed in E.coli. SDS-PAGE result showed that the cloned recombinant protein was expressed in the form of inclusion bodies in the E.coli cell with molecular weight of 16kDa and was amount to 42% of the whole protein in the E.coli cell, which was subsequently dissolved in 7mol/L guanidine chloride and renatured with dilution in refolding buffer containing 0.5mol/L guanidine chloride. In order to obtain pure protein, the renatured boIFN-γ was desalting by Hiprep 26/10 and purified by Hiprep Sephacryl S-200 chromatography. The purified product could inhibit the cytopathic effect, which verified it has the high cytokine activation.