| Interleukin (IL)-22, discovered in 2000, is a member of the IL-10 family of cytokines. The major sources of IL-22 are activated Th1 and NK cells. IL-22 acts via a heterodimeric receptor complex consisting of IL-22R1 and IL-10R2. Neither resting nor activated immune cells express IL-22R1 or respond to IL-22. In contrast, tissue cells at outer body barriers, i.e. of the skin, kidney, and the digestive and respiratory systems are the targets of this cytokine. IL-22 functions by promoting the anti-microbial defense, protecting against damage, and re-organizing non-immune tissues. Furthermore, IL-22 induces acute phase reaction such as serum amyloid A2,haptoglobin,a1-antichymotrypsin. These findings indicate that IL-22 represents a novel type of immune mediator that, although produced by immune cells, regulates tissue protection and homeostasis.1. The cloning and prokaryotic expression of human IL-22Acorrding to the sequence pubblished in NCBI datebase, design the specific primer of human IL-22 mature gene. Use RT-PCR to amplify the human IL-22 non-signal peptide fragment from PHA–activated human peripheral blood T cells. Construct the recombined human IL-22/PQE30 vector by BamHâ… single restriction enzyme digestion, then transformed into M15 strain of Escherichia coli for expression. M15 wxpression strain was propagated in LB culture medium, IPTG was used to induce the expression of recombined hIL-22/His protein. SDS-PAGE demostrated that the recombined hIL-22/His fusion protein was mainly expressed as inclusion body. The cell slurry was disrupted by sonication, and then fusion protein was purified through HiTrap affinity chromatography column after denaturation, refolding and dialysis. The result shows that the purity of IL-22 protein is above 94%. Western blotting showed a single band of molecular mass 18KD. 2. Preliminary study with the biological functions of recombinant human IL-22 proteinThe Gadd45b(Growth arrest and DNA-damage 45 beta),Gadd45g(growth arrest and DNA-damage 45 gamma),SerpinA3(serpin peptidase inhibitor, clade A, member 3) and SAA2(serum amyloid A2) specific Real-Time primer was designed acorrding to the sequence pubblished in NCBI datebase. Use our recombinant IL-22 and commercialized IL-22 as the concentration of 100ng/ml to stimulate the human hepatic carcinoma cell line HepG2 and human colon cancer cell line SW480. Collected cells in the certain time , purificated and reverse transcripted the total RNA of the cells. Use Real-Time PCR to detected the mRNA expression of Gadd45b,Gadd45g,Serpin A3,SAA2 in HepG2 and SW480. The results demonstrated that both our recombinant and the commercialized human IL-22 protein can upregulate the gene expression in the cell lines we detected. This indicates that the human recombinant IL-22 we have obtained is with good biological activity.To sum up, in this study we successfully using prokaryotic expression system to produced the recombinant human IL-22 protein, and preliminary study with the biological activity. In the future, we can use this protein to study its function in tumor. Next, we will do lots of works to further study. |
