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Justine Yeung, Lekha Nair, Lin Sun, Charles Egwuagu; Genetic engineering and characterization of recombinant mouse IL-27 cytokine. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):3489.
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Interleukin-27 (IL-27) is an anti-inflammatory heterodimeric cytokine comprised of IL-27p28 and Ebi3 subunits. It is constitutively secreted by retinal microglia and suppresses intraocular inflammation through endogenous production of anti-inflammatory proteins including IL-10, suppressors of cytokine signaling (SOCS), and complement factor H (CFH). Therefore, targeted delivery of IL-27 into the retina may be beneficial in the treatment of uveitis. However, it has so far not been possible to evaluate the therapeutic efficacy of IL-27 in uveitis or other ocular inflammatory conditions due to the dearth of IL-27. The overall objective of this study is therefore to produce a biologically active IL-27 and to examine whether delivery of exogenous IL-27 can be used to ameliorate uveitis in the mouse experimental autoimmune uveitis model.
The mouse IL-27 (IL-27p28/Ebi3) was engineered by linking mouse IL-27p28 and Ebi3 with an intervening 22 amino acids linker-peptide. The cDNA construct encoding the IL-27 fusion protein was cloned into a pMIB/V5-HIS vector and expression was under the direction of the Baculovirus immediate-early promoter, OpIE2, which allows for high-level, constitutive expression of the proteins in insect cells. The expression vector was transfected into High Five insect cells and stably transfected clones were subjected to several cycles of Blasticidin S HCl selection.
The secreted recombinant IL-27 was partially purified by HIS-TAG affinity chromatography and size-exclusion chromatography using a FPLC system. The recombinant IL-27 has been characterized by Western blot analysis using antibodies against IL-27p28, Ebi3 and the V5 protein tag. Consistent with the predicted molecular size of IL-27, the rIL-27 fusion protein migrated on SDS-PAGE at an apparent molecular size of 54kDa.
A 54-kDa-mouse recombinant IL-27 has successfully been engineered and efficient secretion in insect cell cultures has been demonstrated. Large-scale ex vivo production of the rIL-27 will allow in-depth analysis of this fusion protein and evaluation of its therapeutic potential in murine models of uveitis (EAU) and multiple sclerosis (EAE).
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