Flp-In-293 cell line for expressing bovine gamma interferon and establishment method thereof

Abstract

The invention relates to an Flp-In-293 cell line for expressing a bovine gamma interferon. The cell line is named B1 and contains a gene for encoding the bovine gamma interferon. A method for constructing the cell line B1 comprises the following steps of: constructing preBoIFN-gamma fragments which are correctly sequenced into a recombinant plasmid pcDNA5 / FRT-preBoIFN-gamma-FLAG, cotransfecting Flp-In-293 cells by using the recombinant plasmid pcDNA5 / FRT-preBoIFN-gamma-FLAG and a recombinase expression vector pOG44 by a liposomal transfection method, screening positive clones by a dulbecco's modified eagle medium (DMEM) containing 120mu g / mL Hygromycin B, performing amplification culture, subcloning through limiting dilution analysis, and identifying to obtain a recombinant cell line. The bovine gamma interferon secreted by the cell line B1 has extremely high specific activity and stability.

Description

technical field [0001] The present invention relates to a Flp-In-293 cell line expressing bovine gamma interferon, which contains a gene encoding bovine gamma interferon. The bovine gamma interferon secreted by the cell line has extremely high specific activity and stability, and can be used for clinical treatment of bovine infectious diseases and research on related diagnostic methods. Background technique [0002] Interferon gamma (IFN-γ) is mainly activated by CD4 stimulated by mitogens or specific antigens + Th1 cells, CD8 + Cytokines produced by T cells and NK cells have a wide range of antiviral and antitumor activities and immune regulation functions, such as activating macrophages, increasing the expression of MHC class I and class II molecules, and promoting antigen presentation. They are the body's defense system important parts of. Studies have shown that exogenous IFN-γ can be used not only as a drug for the treatment of infectious diseases or tumors, but a...

AI Technical Summary

Problems solved by technology

[0003] The bovine gamma interferon gene is in a closed state under normal conditions and must be expressed under the stimulation of various specific or non-specific stimulating factors. The traditional method of extracting and purifying bovine gamma interferon from bovine blood is cumbersome and the content Therefore, in order to obtain high-efficiency and cheap bovine gamma interferon, it must be prepared and produced in large quantities through genetic engineering technology, so that its application in animals is possible

The bovine gamma interferon protein expressed in the E. coli expression system cannot undergo post-translational modification and other processes, so its spatial structure and biological activity are quite different from natural antigens; it mostly exists in the form of inclusion bodies or partially soluble, so it can be purified Refolding is difficult; there are many defects such as endotoxin is difficult to remove, so it is limited to a certain extent in clinical application

In the late stage of virus infection in the baculovirus vector expression system, due to cell lysis, the release of intracellular substances will make it difficult to purify the target protein; some hydrolytic enzymes released will also degrade the recombinant protein; there are also certain differences between the processing modification system and mammalian cells

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