Mutant Human Beta Interferon Expression, Purification, And Interferon-like Cytokines Full-length Gene Discovery And Functional Studies

Interferons (IFNs) represent an important class of cytokines having a number of immunomodulatory, antiviral, and antiproliferative effects. IFNs are generally classified into three classes: IFN-α. IFN-β and IFN-γ, each representing protein molecules of different structure. The IFN cytokine family consists of type â… IIFNs (IFN-α and IFN-β) and type â…¡ IFN (IFN-γ). Type â… IFNs were primarily considered as viral inhibitors, whereas type â…¡ IFN, also termed "immune IFN", was generally considered to be uniquely involved in immune reactions. Interferon beta is originally characterized from the supernatants of flbroblasts induced by virus infection. After extensive basic research , IFN-β is now found to be of great therapeutical value in clinic for virus infection, autoimmune disease, and malignant neoplasm. The native human IFN-β is a glycosylation protein, but it is difficult to get the native IFN-β for its complicated preparation and high cost, which restricts the use of IFN-β in clinical trial. With the development of genetic engineering technology, IFN-β has now been recombinantly expressed successfully in E. coil, yeast as well as CHO systems, and has been produced on large scale. Since then, recombinant IFN-β has been studied extensively on basic research and clinical trial. In 1993 IFN-β was proved by FDA for the treatment of multiple sclerosis. It is now used in daily clinical treatment of multiple sclerosis. And it is also used for treatment of chronic viral hepatitis, chronic granulomatous disease, and some malignant tumor. Interferons are also widely studied in our country, but most domestic studies were concentrated on IFN-α. Up to date, IFN-β have not been recombinantly produced yet because of its low expression level and complicated preparation. In the fist part of our present , we have successfully constructed a prokarocytic express vector for recombinant ¹⁷Ser-IFN-β. The recombinant IFN- P was expressed at high level in E.coli and produced on large scale . Moreover, we have recently cloned a novel gene encoding interferon-like cytokine (WN-CK) from human dendritic cell cDNA library by large scale sequencing. Functional analysis of this novel gene is presented in part â…¡ Part â… Expression and Purification of human recombinant IFN-β IFN beta is one of the first new therapeutic products resulting from recombinant DNA technology. Although it has been studied for many years, IFN- P are not proved by National Drug Administration to perform clinical trial in China just for its low amount of prokaryotic expression, the complicated preparation techniques including fermentation, refold and purification Human IFN-³ comprises 165 amino acids and has three cysteine residues, located at amino acid positions 17, 31, and 141. The disu.lfide bond was formed between Cys-31 and Cys- 141, which was necessary for the activity of WN-^. The existence of Cys-17 could affect the formation of correct disulfide bond during protein renaturation. In this study, the codon for cysteine-17 was mutated to serine by the technique of site-specific mutagenesis with a synthetic oligonucleotide primer. A stable and high efficient expression system was established on the basis of the prokaryotic system. The total mRNA was extracted from the cultured human bone marrow stromal cells with polyAltract system. The eDNA was synthesized by reverse transcription with oligo-dT15 as the primer. IFN- P eDNA was amplified by PCR and 17-cysteine was mutated to serine. The...