Studies On The Expression Of Mouse Interferon-λ3 And Its Preliminary Biological Activity

According to the differences of amino acid sequences and receptors, there are three types of interferons (IFNs) such as type-I, type-II and type-III. Type-III interferon family contains three ligands: IFN-lambda1, 2 and 3. IFN-λ3, also called IL-28B , was found and named in 2003 with IFN-λ1 and IFN-λ2. The gene structure of IFN-λ3 is similar to IL-10, but its amino acid sequence is closely related with type-I IFN. Only two subunits of type-III IFNs found in mouse which are IFN-λ2 and IFN-λ3. Mouse IFN-λ3 mRNA can be found in the blood, lung, pancreas, placenta, brain, in mouse. The expression of IFN-λ3 was stimulated by viruses in peripheral blood mononuclear cells, dendritic cells and tumor cells.Since IFN been first found in 1957, researches on IFN have moved forward continuously. Type I IFNs have been approved for use in multiple clinical indications, however, some adverse side effects such as fatigue, fever were reported. Owing to different biological activities of IFN-λs and the expression level and tissue distribution of IL-28R relative to the type I IFNs and their receptor, IFN-λs may serve as a new alternative therapeutic choice to type I IFNs.In this research, cDNA encoding mouse IFN-λ3 was successfully cloned and was expressed in both prokaryotic and eukaryotic system. The biologic functions of mIFN-λ3 were also investigated. All of these studies construct the foundation for further research. The main results of this paper are as follows:Firstly, to express mouse mature IFN-λs protein, we have constructed an Escherichia coli expression systerm for rapid expression of the mIFN-λs gene. The procedure entails cloning mIFN-λ3 gene into the pET-44 vector, which allowed expression of mIFN-λ3 with a His-tag.Secondly, to construct the eukaryotic expression system of mIFN-λ3, a pair of specific primers were designed. The cDNA encoding mIFN-λ3 was successfully cloned by polymerase chain reaction (PCR) from a purchased expression clone EX-Mm18539-M03. The mIFN-λ3 was cloned into pcDNA3.1(+) vector. The DNA sequence of the insert was identical to the published sequence encoding the full length of mIFN-λ3.Thirdly, the constructed recombinant vector pcDNA3.1-mIFN-λ3 was transfected into 293 cells. MTT assay was performed to analyze the influence of the target protein on the viability of A549 and SW620 cells. Moreover, we investigated whether recombinant mIFN-λ3 protein induces these cells to produce MxA protein. Results: The mIFN-λ3 protein was stably and consistently expressed in 293 cells. The protein inhibited the proliferation of A549, SW620 cells and induced the expression of MxA in these cells. We found that mIFN-λ3 protein is able to induce the production of MxA related to antivirus in cells.In conclusion, the cloning and expression of both prokaryotic and eukaryotic recombinant mIFN-λs gene, the researches on mIFN-λ3 biologic activity provide a fundamental basis for further study of theoretic investigation and clinical appalications of mIFN-λ3.