Molecular Characterization Of IFN-γ In Tree Shrew And Construction Of CHO-S Monoclonal Cell Line With Lentivirus Expression Vector

Objective(s): As a new experimental animal of non-human primates,tree shrew has been used to develop animal models of a variety of human diseases.In immunological research,antibodies and cytokines are key experimental tools,which can be used to evaluate immune function and discover new mechanisms,but due to the lack of commercial reagents and standardized detection procedures,it hinders the further research and application of tree shrew as an experimental animal.Interferon gamma(IFN-γ)is a cytokine that plays an important role in both innate and acquired immune responses,and is indispensable for immune detection.Because the IFN-γamino acid sequence of tree shrew is relatively conservative,specific and different from human and main experimental animals,no substitute has been found for crosssharing.At present,most tree shrews recombinant cytokines are mainly expressed through prokaryotic cells,and their immunogenicity is affected because of the lack of post-translational modification expressed by eukaryotic cells.Therefore,the eukaryotic expression of recombinant tree shrew IFN-γ using CHO cells will provide a better reference for the research and application of tree shrew model,and also provide convenience for the development of specific antibodies and cytokine detection.Methods: The IFN-γ gene of tree shrew was amplified by PCR,and the mammalian cell expression plasmid was constructed and expressed.The expression was detected by Western Blot and Coomassie staining.The lentivirus was prepared by packaging lentivirus expression system,and then infected with HEK293 cells and CHO cells.The expression conditions were explored and optimized,the single cell lines with high expression were screened,and the scheme with the highest economic efficiency was selected to complete the expression of the recombinant protein.After optimizing the process,a large number of tree shrews IFN-γ were expressed,and the recombinant tree shrew IFN-γ was purified by nickel column affinity chromatography.Results: The main results were as follows:(1)the transient mammalian expression plasmid was constructed successfully,and a small amount of expression was detected in 293 T,293FT and 293 F.(2)the lentivirus plasmid expressing IFN-γof tree shrew was successfully constructed and the lentivirus with high titer was prepared.The lentivirus was used to screen the single cell strain with high expression in CHO-S cells.After the verification of Fed-Batch technology,the recombinant tree shrew IFN-γ with purity more than 90% was purified.Conclusion(s): The expression efficiency of stable cell lines obtained by lentivirus infection was better than that of transient transfection expression;the lentivirus expression system constructed quickly screened the tree shrew IFN-γ highdose single cell line;Fed-Batch cell culture prolonged the cell culture time and increased the protein yield.