Expression And Purification Of IL-37and Its Role In Inhibiting Collagen-induced Arthritis

ObjectiveInterleukin(IL)-37is a new member of IL-1family. It has five different subtypes (IL-37a-e).Some studies have shown that IL-37b has anti-inflammatory effects in various diseases. IL-37canbe secreted into the extracellular and binding to the receptor, then regulating cell activity. When IL-37was transported into the nucleus, it can be used as transcription factors to regulated geneexpression. IL-37shares significant sequence homology with IL-18. IL-37can bind to IL-18binding protein. The activity of IL-18was reduced by IL-37. New research found that IL-37increased in rheumatoid arthritis synovial tissue.Rheumatoid arthritis is an autoimmune disease that results in a chronic, systemicinflammatory disorder. It can be a disabling and painful condition. Therefore, rheumatoid arthritishas brought great pain for patients. It affected the quality of life of patients seriously. Thepathogenesis of rheumatoid arthritis has necessarily linked with a variety of inflammatorycytokines. Because IL-37has anti-inflammatory effects, this study intends to build IL-37eukaryotic and prokaryotic expression vector and purify the protein, and construct the lentiviral IL-37vector. Then, we use purified proteins and collected lentivirus to explore the impact of IL-37oncollagen-induced arthritis.Methods1. Total RNA was extracted from human skin tissue. Full-length coding sequence of IL-37was amplified by RT-PCR. The cDNA was cloned into pcDNA3.1(+) vector. Eukaryoticexpression vector of IL-37was designated as pcDNA3.1(+)-IL-37.2. The coding sequence of mature IL-37was amplified with pcDNA3.1(+)-IL-37as atemplate. The sequence was cloned into pET-44vector. Prokaryotic expression vector of IL-37was designated as pET-44-IL-37.3. The expression of IL-37was induced by IPTG. IL-37protein was purified with His-tag,and endotoxin was removed. 4. Full-length coding sequence of IL-37was amplified by PCR with pcDNA3.1(+)-IL-37as atemplate. The sequence was cloned into pLVX-IRES-Green1vector. The lentiviral vector of IL-37was constructed. To package and collect lentiviral, the vector was transfected into293T cell lines.5. To study the effect of IL-37, collagen-induced arthritis was constructed. The IL-37proteinand lentiviral was used to investigate the role of IL-37in inhibting collagen-induced arthritis.Results1. Total RNA was extracted from human skin tissue. A657bp IL-37gene sequence wasamplified by RT-PCR. IL-37eukaryotic and prokaryotic expression vectors were constructedsuccessfully.2. IL-37protein was expressed successfully. After purification and removal of endotoxin,pure IL-37protein was obtained. We found that the purified IL-37protein can inhibit theinflammation of blood cells, which explains that the purified protein has biological activity.3. Lentiviral vector of IL-37was constructed successfully, and was packaged to obtain thevirus particles with high titers. The virus particles were contagious and expressed IL-37mRNA ineukaryotic cells.4. A model of collagen-induced in DBA/1mice was replicated successfully. Virus particlesand protein of IL-37were injected into mice at the tail vein and peritoneal respectively. We foundthat IL-37has a role to alleviate joint inflammation in mice.ConclusionWe had constructed the eukaryotic and prokaryotic expression vector and Lentiviralvector of IL-37, and purified the IL-37protein. IL-37protein has been proved that hasbiological activity. The lentiviral particles can infect eukaryotic cells and express IL-37mRNA. We used the protein and virus to explore the effect of IL-37in collagen-inducedarthritis. We found that IL-37can inhibit the development of the disease in mice.