The Study Of The Preparation And Antitumor Activities Of Recombinant Human IFN-λ1

IFN-λS (including IFN-λ1, IFN-λ2, and IFN-λ3) is a newly identified IFN family whose characters related to the type I IFNs and IL-10family members. IFN-λs act through a cell surface receptor composed of two chains, the first one (CRF2-12/IFN-λR1/IL-28Ra) being1FN-λ specific and the second one (CRF2-4/IL-10R2) shared with IL-10. IFN-λs signal through the IFN-λR1and activate JAK-STATs pathways, as same as type I IFNs. So, IFN-λs exhibit several common features with type I IFNs:antiviral activity, anti-proliferative activity and in vivo antitumor activity. It is noteworthy that, the expression of IFN-λR1(IL-28Ra) is tissue-specific, and there is very low expression in the bone marrow and brain spinal cord. This feature enables IFN-λs to be a new potential IFN therapy reagent. It can be effective in reducing fever, depression and bone marrow suppression and other side effects, compared to IFN-α.In our laboratory, we have constructed and expressed the new recombinant human IFN-λ1(rhIFN-λ1), and did some preliminary study of bioactivity of rhIFN-λ1. In the previous experiments, it was indicated that rhIFN-λ1could activate the JAK-STATs pathways in HeLa cell, and could inhibit the proliferation of the specific cells. There are some problems waiting for overcoming such as how to increase the recombinant protein's expression level in Pichia pastoris yeast and low-purity with existing purification procedure. The present study focused on the expression, purification, identification of physical and chemical properties and antitumor activity of rhIFN-λ1, also do some related experiments.Firstly, BMMY medium was used for efficient expression of rhIFN-λ1in Pichia pastoris yeast, and then SP FF cation exchange chromatography and Superdex75Size-Exclusion chromatography was used to purify the recombinant protein, and obtained high purity of rhIFN-λ1. After that, we identified the molecular weight, the N-terminal sequencing, isoelectric point, disulfide bonds, and other physical and chemical properties of the recombinant protein. Then, in this part our research works mainly focus on the anti-tumor effect of rhIFN-λ1in vitro and in vivo. We used qRT-PCR and Western blot to determine the expression of IFN-λR1in HT29and HCT116cell lines. The results showed that both cell types expressed the IFN-λR1receptor genes with variable transcriptional and protein levels. The MTT analysis showed that rhIFN-λ1inhibited the proliferation of HCT116cells in a dose-dependent manner, but with a less efficacy in HT29cells than HCT116cells. rhIFN-λ1also activated the STATs and induced apoptosis in both types of cells. In the in vivo study, we found that rhIFN-λ1suppressed tumor growth in a dose-dependent fashion, with an inhibition rate of52%of HCT116(P<0.01) and56%of HT29(P<0.01). Immunohistochemical staining in the sections of xenografts with Ki-67expression were also detected and the result demonstrated that the percentage of positive staining in cells gradually decreased with the increase in the concentration of rhIFN-λ1and showed statistically significant differences.Secondly, Mutagenesis with overlap PCR technology was used to construct multi-copies expression vector of IFN-λl-Nm (IFN-λ1N46Q). The6copies expression vector of IFN-λ1-Nm (pAO815-6×IFN-λ1-Nm) was then transformed into Pichia pastoris and IFN-λ1-Nm was expressed by secreted model. The SP FF and Superdex75were used to purify the mutant interferon. The biological activity of IFN-λ1-Nm was detected by examining the expression of HLA-ABC and dual luciferase reporter gene analysis, and the results showed that there are no significant differences between Mutations before and after. On this basis, we are also did the acute toxicity experiments. The result indicated that there are no side effects, which establish a solid foundation for the future experiments.In conclusion, rhIFN-λ1is a potent reagent and can be selected as an anti-tumor agent and in fact it has been demonstrated to have anti-tumor effects in two types of colon tumor cells. This is the first report that shows the in vivo rhIFN-λ1inhibition in human colorectal tumor growth alone, and it provides strong evidence to indicate that rhIFN-λ1may be developed into a more promising therapeutic reagent for the treatment of tumors in the future.