Cloning And Prokaryotic Expression Of Tupaia IFN-gamma Protein

Objective1. Cloning tupaia interferon gamma gene.2. Construction the prokaryotic vector of tupiaia interferon gamma, and expressingtupiaia IFN-gamma protein in Escherichia coli of BL2(DE3) PlysS.MethodsThe tupaia interferon-γ gene was amplified by RT-PCR (reverse transcriptionpolymerase chain reaction) from total RNA of tupaia PBMC. Then, the gene was connectedinto the vector of pRSET-B to construct the prokaryotic express vector of IFN-γ-pRSETB.The vector of tupaia IFN-γ-pRSETB was expressed in Escherichia coli of BL2(DE3) PlysSby IPTG inducing.ResultsThe fragment of interferon gamma was obtained from the PBMC of tupaia byRT-PCR.The coding sequence of tupaia interferon gamma gene contain509nucleotides.The corresponding amino sequences of interferon gamma between tupaia and human sharedhomologies of63%.The fragment of coding sequences of interferon gamma was connected with the vectorpRSET-B. The recombinant vector was confirmed by direct sequencing,the prokaryoticexpress vector IFN-γ-pRSET-B was constructed successfully.The IFN-γ-pRSET-B vector was induced by IPTG in Escherichia coli of BL2(DE3) PlysS.The recombinant protein of IFN-gamma could be expressed in the form of inclusion bodies. The recombinant protein was confirmed at the position of18KD by western blot.