Bioactivity Analysis And Expression Of PIFN-γ And PIL-18 Genes In Mammalian Cells

IFN-γis a kind of cytokine, which plays a very important role in resistance to viruses, bacteria, parasite, tumor and in immunomodulatory activities. Interleukin-18 is a new cytokine, known as interferon-γ-induced factor (IGIF)-because of its ability to intensively induce IFN-γproduction. Furthermore, its function is similar to IFN-γ. At present, many studies have indicated they play a very important role in treating viral diseases and are predicted to be used as a new adjuvant.In the medical field, using genetic engineering has successfully expressed many kinds of cytokines in protokaryon Bacillus coli, eukaryon yeast system and mammalian cells, which will make possibility of large-scale production of recombinant cytokines. So the study has been designed to search for and set up the cell strains of PIFN-γand PIL-18 by use of mammalian cell system, which will establish foundation for large-scale production and application. We have acquired the results below.Firstly, using pGEM-PIFN-γand pGEM-PIL-18 as templates were amplified by PCR. Then the fragments and PEGFP-N1 vector were digested with two enzymes, which were ligated together and transformed into Ecoli cells finally. The results indicated that PIFN-γand PIL-18 genes were correctly inserted into PEGFP-N1 vector by means of PCR, enzymes digestion and sequencing.Secondly, the recombinant plasmids pEGFP-PIFN-γand pEGFP-PIL-18 were respectively transfected into BHK cells and CHO-K1 cells by Lipofectamine. Fluoroscopy, RT-PCR, SDS-PAGE and Western-blot were used to detect and identify the expressed products in mammalian cells; the bioactivity of the products were detected by MTT assay. The results indicated that the green fluorescence could be observed in the cells transfected with pEGFP-PIFN-γand pEGFP-PIL-18. Furthermore, the interest genes were amplified and the specific proteins were detected and possessed obvious bioactivity.Thirdly, after 24h pEGFP-PIL-18 was identified through screening with G418, then obtained subcloned cells through limited dilution method, detected expression conditions with techniques of fluoroscopy and RT-PCR. The results indicated stable CHO-K1 cell line of PIL-18 was established.To Sum up, the PIFN-γand PIL-18 genes has successfully been expressed in mammalian cells at the first time, which can be utilized to develop the biological products and be served as a new product in the control of porcine diseases.