Cloning, Expression, Bioactivity Study And Preparation Of Monoclonal Antibody For Porcine Interleukin-22

Interleukin(IL)–22 is a member of the IL-10 cytokine family that is primarily secreted by T lymphocytes and innate lymphoid cells. IL-22 acts via the signal transduction in immune response by heterodimeric receptor complex consisting of IL-22R1 and IL-10R2. Recently growing studies from murine or human IL-22 demonstrate that IL-22 expression has been associated with Anti-inflammatory and pro-inflammatory immunity. IL-22 primarily target mucosal epithelial cells, play a vital role in the repair of mucosal wound, inflammatory response, and fight against infection.While, the influence of IL-22 long-term overexpression on tumor cell growth in vitro and tumor formation and progression in vivo has been investigated in several studies. Intestine as an important place for the host immune response, IL-22-mediated barrier maintenance involves support of the epithelial lining’s structural in-grity as well as its functional activation. Regarding IL-22, recently research are focused on human and murine, a few reports of fish IL-22 also had been found, However, as we know, there is no report of porcine IL-22 until now. In this study, we designed to identified whether the porcine IL-22 protectting the host from inflammation or pathogen invasion, lay the foundation for the research of the pig IL-22.(1) Cloning, expression and bioactivity studies of porcine interleukin-22.We amplified swine IL-22 fragement about 586 bp and mature fragement about 476 bp from the peripheral blood induced DC cells of swine, and constructed prokaryotic PET-30 a expressing vector. BL21(DE3)bacteria transformed with IL-22 pET-30 a was successfully expressed IL-22 protein following IPTG induction, about 22 KD. IL-22 was purified through Ni-NTA affinity chromatography.The IPEC-J2 up-regulated both interleukin-18(IL-18), beta-defensin-2(BD-2) and interferon(IFN)-λ expression after being treated by recombinant purified porcine IL-22. When the mature IL-22 stimulate IPEC-J2 cells at the final concentration of 0.04μg/mL, Increasing levels of three Cytokine were accumulated with the prolonged of stimulation time, at 48 h, the quantity were maximized.Additionally, our experiments exhibited a role for IL-22 in IPEC-J2 cells resistance to PEDV-CV777 and PoRV infection. These results demonstrated that our porcine recombinant IL-22 has bioactivity, which can inducing IL-18、IFN-λ and BD-2 production, and playing some role in antiviral defense on the swine IPEC-J2 cells. This providing a foundation to further research the role of porcine IL–22 in mucosal immunity.(2) Preparation Monoclonal Antibodies against porcine IL-22.Splenocytes from 6-8 weeks BALB/C mice immunized with purified target protein, which was obtained from the first half study, were fused with SP2/0 cells to produce MAbs, which were selected with indirect ELISA. MAbs(2G10, 2C7, 6B11 and 7F3) belonged to IgM K subtype were produced. Supernatant and ascitic of the monoclonal antibodies 6B11 reacted with mature IL-22 protein in ELISA at a titer of 1︰ 1600 and 1 ︰ 51200, and the monoclonal antibodies 7F3 in ELISA at a titer of 1︰100 and 1︰6400. This research supplied important tools for detecting and studying IL-22.