Expression Of Recombination Human IL-13 In Pichia Pastoris And Its Effect On The Induction Of DC From

Postgraduate : Jiang Yang Tutor : Zhang XueguangInterleukin-13 (IL-13) identified in 1993 is a pleiotropic cytokin secreted by activated TH2 cells. IL-13 shares a variety of biologic functions with IL-4, such as anti-inflammatory effects on monocytes and macrophages, regulation of B cell function and adhesion molecule expression on endothelial cells, as well as inhibition of monocyte differentiation and B cells. It also can inhibit human immunodeficiency virus type 1 production in primary blood-derived human macphages in vitro and suppress the growth of some carcinoma. IL-13 has important function during the processor of many disease, especially the allgery illness of asthma. More and more attention has been payed to its application in lab and in clinic.In order to obtain a high yield of recombinant human IL-13 , we cloned the gene for IL-13 and expressed it in Pichia pastoris . Then we investigated the biological activities of rhIL-13 in vitro, especially its effect on the induction of DC from PBMC.Part I Expression of Recombinant Human IL-13 in Pichia pastorisIn order to obtain a high level production of rhIL-13, an artificial gene with the optimal codon usage of Pichia pastoris was synthesized for rhIL-13. Eleven oligodeoxynucleotidefragments, 14 to 66 base in length , were first linked into two small fragments of around 140~200 bp with T4 ligase. The small fragments were then amplified by polymerase chain reaction (PCR) with Taq DNA polymerase, and linked to each other to form the full-length artificial gene in a second round of PCR using Pfu DNA polymerase .The amplified DNA fragment was digested with Xholand XbaI, and ligated into the cloning vector pSK. The DNA sequence of the insert was analyzed on an automatic DNA sequencer. The artificial gene encoding rhIL-13 was cleaved with Xhol and Xbal from the cloning vector after the sequence had been verified, and recloned into the predigested yeast expression vector pPICZA, which carries an a factor secretion signal sequence with a Zeocin marker for antibiotic selection. The plasmid was then linearized with Sad and introduced into Pichia pastoris by electroporation. Colonies that grew on the YPD/Zeocine plates were tested for expression after growth in a 2ml of BMGY medium, followed by cultivation on BMMY medium. The recombinant protein secreted by the yeast was analyzed by 15%SDS-PAGE, ELISA and Western Blotting;a yield of 20mg/L was obtained in a 3. 7L fermentor. Using routine fermentation methods followed by ion chromatograph -> gel chromatography and FPLC purification of the fermentation production, purifed rhIl~13 was obtained.Part II Biological activity of rhIl-13 and its effect on the induction of DC from PBMCThe biology activity of the rhIL-13 in yeast supernatant is the same as that of standard produced by E coli determined by stimulating the proliferation of B9-11. IL-13 shares a variety of biologic functions with IL-4. In the present study, we compared IL-13 and IL-4 in their abilities to induce DC differentiation andfound that adherent PBMC cultured in GM-CSF , TNF a and IL-13 displayed morphological characteristic of DC generated in media containing IL-4. They formed cellular clumps and had extensive dendrites sharp. Fluorescence-activated cell sorter analysis showed that they expressed a high level of MHC class II -. GDI a, CD80, CD83, CD86. The purity and yield of both DC populations are not significantly different. The phagocytosis of immature DC induced by IL-13 is potent. They were also equally potent stimulators of allogeneic lymphocytes in the mixed lymphocyte reaction. RhIL-13 can substitute for IL-4 in the proliferation and development of dendritic cells.