Expression And Bioactivity Study Of Interleukin-7 Splice Variant Lacking Exon 4 And 5

Background:A variety of cells in body can produce IL-7, the main biological role of IL-7 is involved in the body’s immune regulation; IL-7 has a lot of splice variants, and the research of IL-7 has only just begun. It is reported that IL-7 has a strong anti- fibrosis, the mechanism of it is mainly through activation of JAK1/STAT1 signaling pathway and down regulate the expression of Smad7 expression of GF-β1 to block TGF-β-induced collagen synthesis.IL-7δ4/5, the IL-7 splice variant lacking exon 4 and 5, and the research of it has just begun, Whether it also affects the development of pulmonary fibrosis also needs to be researched. Objective:In this study, we adopt the prokaryotic expression vector p ET21 b to express rhIL-7δ4/5, dissolving, refolding and purification the inclusion body,then get the protein with renaturation and high purity. Using the technology of Western Blot to identify the protein, using TGF-β1 and IL-7 as a comparative to analysis its impact on lung fibroblasts MRC-5, and to explore its mechanism. Methods:1.Preparation of rhIL-7δ4/5 protein: Use 18-T-IL-7δ4/5 which preserved in o ur lab as the template to carry out the TA cloning, and construct a prokaryotic expression vector pET-21b-IL-7δ4/5successfully. Add inducer IPTG into the rhIL-7δ4/5 to induce its expression.Then analyze its solubility, and optimizes its induced concentration and induction time. The IL-7δ4/5 protein was refolded with gradient dialysis and purified with affinity chromatography chromatography. After purification,we use the technology of Western Blot to identify the protein of IL-7δ4/5.2. The role of the rhIL-7δ4/5 protein on the lung fibroblasts MRC-5 cell: MRC-5 cells, which is cultured in vitro were irritated with IL-7, IL-7δ4/5 and TGF-β1, use the method of MTT to detect the proliferation of MRC-5 cells. And use the method of Western blot to analyze the expression of collagen I and α-SMA protein. Restuls:1.The inducible expression, purification and renaturation of rhIL-7δ4/5 protein: We construct the recombinant p ET-21b-IL-7δ4/5 plasmid. Then use the technology of PCR to detect the amplify fragment sizes, the result is as the same as IL-7-δ4/5(345bp) that expected and the result of DN A sequencing is also as the same as that of GenBank reported. Cultivate the positive clones that recombinant plasmid was transfection successfully, then induce it with IPTG, the result of SDS-PAGE electrophoresis shows the inducible expression is successful. The isolation and purification of inclusion body was dissolved, dilute and Gradient dialysis, and the refolding efficiency of IL-7δ4/5 was about 45%, the purity of IL-7-δ4/5 was﹥95%. Finally we use the technique of Western Blot to identify it.2. The role of the rhIL-7δ4/5 protein on the lung fibroblasts MRC-5 cell: Use TGF-β1, rhIL-7 and rhIL-7δ4/5 to irritate MRC-5 cells, The result is that TGF-β1 could promote cell proliferation(P <0.01), and the collagen I、α-SMA expression in cells increased(P <0.01) significantly induced. IL-7 was showed inhibition of cell proliferation, reduce the role of α-SMA protein expression, while the proliferation of IL-7δ4/5 neither influence the collagen I and α-SMA protein expression nor influence the proliferation of MRC-5 cells. This suggests that IL-7δ4 /5 for pulmonary fibrosis has no effect.