Preparation Of Recombinant Human Interleukin-38 And Its Inhibition Of Bleomycin-induced Pulmonary Fibrosis In Mice

Objective: To clone the interleukin(IL)-38 gene, construct its eukaryotic/prokaryotic expression vector, purify the recombinant protein, and test its biological activity. To construct the lentiviral vector carrying IL-38 gene, pack its virus particles, and study the therapeutic effects and its mechanism of IL-38 on the pulmonary fibrosis induced by bleomycin(BLM) in mice. Methods: The total RNA was extracted from human skin tissue and the IL-38 gene was cloned into pcDNA3.1/V5-His-TOPO to construct its eukaryotic expression vector pcDNA3.1-hIL-38-V5-His by RT-PCR. This vector was taken as template for PCR amplification to construct its prokaryotic expression vector pET-44-hIL-38. The prokaryotic expression plasmids were transformed into host E. coli BL21(DE3). Then, IPTG was used to induce expression of target IL-38 protein with 6×Histag in its C-terminal. The recombinant protein was purified through Ni2+-chelating affinity chromatography by 6×His-tag. The activity of purified protein was analyzed by stimulating LPSinduced THP-1 cells. The lentiviral vector pLVX-hIL-38-IRES-Green1 carrying IL-38 gene was constructed and the lentiviral particles were packaged by co-transfection 293 T cells with the recombinant expression vector and three packaging vectors. The pulmonary fibrosis of C57BL/6 male mice was induced by intranasal bleomycin administration. The IL-38 virus supernatant was administrated intranasally to explore the effects on the occurrence and development of pulmonary fibrosis in mice. Results: The sequences of vectors, which were constructed in this study, were consistent with genetic sequences in GenBank. The IL-38 protein induced by IPTG was mainly in the form of soluble pattern, and the purity was above 98% explained by SDS-PAGE gel electrophoresis and Western blot analysis. The purified protein has a high biological activity through the cell stimulation experiments. Animal experiments results showed that IL-38 may reduce lung infiltration of inflammatory cells, deposition of collagen and extracellular matrix in cells; improve the pulmonary alveolar wall structure; inhibit the production of tumor necrosis factor(TNF)-α, IL-4, IL-6, IL-17, monocyte chemotactic protein(MCP)-1, macrophage inflammatory protein(MIP)-2, granulocyte-macrophage colony stimulating factor(GM-CSF), and other pro-inflammatory cytokines and pro-fibrotic cytokines, promote the production of antifibrotic factor IFN-γ. Conclusion: In this paper, IL-38 was cloned into the eukaryotic, prokaryotic, and lentiviral expression vectors, bioactive IL-38 protein was prepared and IL-38 lentivirus was packaged successfully. The lentivirus-mediated IL-38 can inhibit the development of pulmonary fibrosis induced by BLM in mice. Our results provide a theoretical foundation for further research and clinical application of IL-38.