Expression, Purification, And Identification Of Mouse Interleukin-17 And Preparation Of Anti-mouse Il-17 Antibody

Objective: Through genetic engineering techniques obtain the active recombinant mouse IL-17 (recombinant mouse Interleukin-17, rmIL-17) protein to detect the activity of rmIL-17 certification and immunogenicity.At the same time with the purified recombinant protein rmIL-17 mice and rabbits,and identification of anti- rmIL-17 monoclonal antibody and polyclonal for further study of the biological function and its mechanism of IL-17 provide the basis. Methods: mouse Interleukin-17 gene was amplified in vitro by RT-PCR, then, the target gene was cloned into prokaryotic plasmid Pet32a(+).After the recombinant plasmid was transformed into E.coli BL21, it was expressed after induction with IPTG. Passed through affinity chromatography column to get the purified IL-17, New Zealand rabbit were immunized with rmIL-17 and polyclonal antibody was produced. The immunogenicity was identified by Western blot and ELISA. With rmIL-17 protein in mouse fibroblasts in vitro stimulation experiments, 3T3 mouse fibroblasts will adjust the density of 4×105/ml in 24-well plate, rmIL-17 protein were added to the final concentration 0.01,0.1,1, 10,100, 1000ng/ml, were incubated 24 hours in a humidified CO2 incubator, take the supernatant, with the mouse IL-6 ELISA kit Quantitative ELISA detection of IL-6 concentration. Under the same conditions to 1000ng/ml recombinant extramembranous protein 29KD of Schistosoma japonicum as a negative control, medium as a blank control; with rmIL-17 protein in neutrophil recruitment in vivo experiments, the experimental group with sterile PBS dilution of 0.5μg rmIL-17 protein to 500μl, BALB / C mice intraperitoneally. Intraperitoneal injection of equal amount of recombinant extramembranous protein 29KD of Schistosoma japonicum to do outside the control group. 0.5ug rmIL-17and 10ug anti-mouse IL-17 neutralizing antibody diluted with sterile PBS to 500ul,37℃after 15 minutes incubation,intraperitioneal injection,as the middle groups. After 4 hours, mice were killed by blood letting, open abdominal cavity with 2.5ml of PBS containing 3mM EDTA washing, washing up liquid collected, the total number of cell counts and neutrophil staining; Simultaneously, 6-8 week-old female BALB/c mice were immunized three times with purified rmIL- 17, then the spleen cells of the immunized mouse and SP 2/0 cells were mixed, and fused by 1 ml of 50% PEG 4000. The hybridoma cells were cultured in the HAT selective medium. The supernatants secreted from the hybridoma cells were detected by ELISA 10 to 15 days later. Then the McAb in the supernatants was identified by the analysis of the isotype,. Western-blotting experiments identify immunological characteristic of monoclonal antibodies. Results: The recombinant plasmid pET32a-mIL17 and expressed in E. coli. Column had been purified rmIL-17 protein, prepared polyclonal antibody anti-rmIL-17 Western-blot experiments confirmed the good immunogenicity; in vitro cell stimulation experiments showed that the protein can stimulate the mouse 3T3 fibroblast cells to produce IL-6(Interleukin-6) and has a dose-effect relationship; With the accession of rmIL-17 protein concentration , 3T3 mouse fibroblasts the amount of IL-6 also will increase; the cells Recruitment experiments showed that purified rmIL-17 protein has a role in the recruitment of neutrophils. recombinant extramembranous protein 29KD of Schistosoma japonicum as the control group to anti-mouse IL-17 neutralizing antibody and in control group (0.481±0.154)×106 and the experimental group (4.27±1.10)×106 difference was significant (P <0.05), and in group (2.01±0.90)×106 and the experimental group (4.27±1.10)×106 difference is also significant (P<0.05); Experiments confirmed the in vitro activity of rmIL-17 .Preparation of the anti-rmIL-17 monoclonal antibodies.2 monoclonal antibody subtypes were IgG1, IgG2a; Western blotting confirmed the two immunological characteristics of monoclonal antibodies. Conclusion: Mouse IL-17 gene was successfully expressed in vitro, and the recombinant protein has significant biological activity. rmIL-17 polyclonal antibody and monoclonal antibody preparation for our next experiment laid the foundation.