Prokaryotic Expression Of Mouse Interlukin-33 And Preparation And Applications Of Its Polyclonal Antibody

Interleukin (IL)-33, also called IL-1F11, first described as a proinflammatory cytokine in 2005 through a computationally derived profile of members of the IL-1 cytokine family, belongs to IL-1 family, and is most closely related in structure to IL-18 and IL-1β. IL-33 mRNA is broadly expressed in many tissues of human and mouse, but it is more restricted at the level of cell types. High levels of mouse IL-33 mRNA can be found in the stomach, lung, spinal cord, brain, skin, resting dendritic cells and activated macrophages in mouse. Human smooth muscle cells and epithelial cells forming the bronchus or small airways show a constitutive expression of IL-33 mRNA. IL-33 mediates its biological effects via ST2, activates transcription factor NF-κB and mitogen-activated protein (MAP) kinases, and drives production of T helper type 2 (Th2)-associated cytokines and thereby promotes defense and pathology in immune and inflammatory diseases.Studies on function of IL-33 are still on the way. In this research, cDNA ecoding mouse IL-33 (mIL-33) was successfully cloned and was expressed in Escherichia coli. Rabbit anti- mIL-33 polyclonal antibody was identified, further more, the distribution of mIL-33 protein in various normal tissues and some inflammatory tissues and the roles of IL-33 in different diseases were also discussed. All of these laid the foundation for further research. The main results of this paper are as follows:Fistly, to construct the recombinant vector carrying IL-33 cDNA, a pair of specific primer was designed according to the published mIL-33 sequence searched from GenBank (No. AY905582). With the specific primer, cDNA encoding mIL-33 was successfully cloned by reverse transcription-polymerase chain reaction (RT-PCR). The cDNA was cloned into pcDNA3.1(+) vector. The DNA sequence of the insert was identical to the published sequence encoding the full length of mIL-33.Secondly, to express mouse mature IL-33 protein, an Escherichia coli expression systerm for rapid expression of the mIL-33 gene was developed. It involved of cloning IL-33 gene into the pET-44 vector, which allowed expression of IL-33 with a His6-tag. The expressed IL-33 fusion protein was purified by Ni-NTA affinity chromatography. The purified mIL-33 appeared as a single band on SDS-PAGE and the purity was more than 95%. The potency of purified mIL-33 in inhibiting P815 cell survival was stronger than that of commercial mIL-33.Thirdly, polyclonal antibody of mIL-33 protein was prepared. ELISA analysis showed the antibody with high titer of 1:32000. Western blot analysis showed this antibody could specifically recognize mIL-33 protein. Immuhistochemistry staining estimated that mIL-33 protein may be located in cytoplasm or nucleus of bronchial epithelial cells and liver cells.Fourthly, anti-IL-33 antibody was evaluated against articular inflammation in a murine model of rheumatoid arthritis.In conclusion, the cloning and expression of recombinant mIL-33 gene, the identification of anti-IL-33 antibody and the discussion of the relationship between IL-33 and some inflammatory and immune diseases provide a fundamental basis for further study of theoretic investigation and clinical appalications of IL-33.