The Preparation And The Effects Of Anti-Dermatitis Of Antibody Against Recombinant Human Interleukin-31

Objective: To express high-level recombinant human IL-31 for the preparation of anti-IL-31 antibody (polyclonal antibody/monoclonal antibody). Investigating the antagonism of anti-IL-31 antibody in dermatitis lay the foundation for the development of new drugs. Methods: (1)E. coli BL21(DE3) was raised to express recombinant human IL-31 protein induced by IPTG. Collecting thallus after centrifuging broken by ultrasonic. The inclusions were dissolved in urea and purified by Ni-NTA agarose gel column. The recombinant protein was quantitative packaged. (2) Preparing polyclonal antibody by immunizing health rabbits with the immunogen, this was rhIL-31 emulsifying with the identical volume adjuvant. It was determined by the indirect ELISA and Western blot. (3) Dividing IL-31 PcAb into 3 groups: high dose group (40μg), medium dose group (20μg), and low dose group (10μg), and then treat the dermatitis Balb/c mice through subcutaneous injection and the direct application onto the skin. Set the negative control group (Non-treatment group) and blank control group. We got the Paraffin-embedded sections of skin stained with HE, and observed the changes in histological changes, appearances and behaviors of the mice on the third day after the treatment. We got the quantitative detection of serum IL-6, IL-8, MIP-3βand L-selection. (4) Preparing polyclonal antibody by immunizing health rabbits with the Synthesize peptide which was cross-linked to bovine serum albumin (BSA). Purify the antibody by protein an affinity chromatography identified by ELISA and Western Blot. Results: We got the high-level expression of rhIL-31 identified by SDS-PAGE. There was the peak titer after 4W by Immunization, and the high level can be stabilize after 5～6W .The titer was 1:15000～1:20000 detected by indirect ELISA and was 1:16 by double agar diffusion. IL-31 PcAb Antibody purified could specifically recognized rhIL-31 detected by Western Blot. HE stain showed the infiltration of inflammatory cells of the subcutaneous and intradermal in Treatment Group, and a certain degree of dose-dependent. Compared with the negative control the infiltration decreased significantly. Serum levels of ELISA detection showed serum IL-6,IL-8 in treatment Group were significantly lower than negative control group (p<0.05) . Compared with the two-ways in one-dose there is no significant difference between the two-ways. Serum L-selection in treatment group was significantly lower than the negative control group and higher than the blank control group. There was no significant difference by the statistical analysis (p>0.05) . Averages of serum MIP-3P in treatment group was lower than the negative control group (p<0.05) . Compared with the two-Ways in one-dose there is no significant difference between the two-Ways. The titers of ascites induced by two strains hybridoma cells were 1:10000(8A1)and 1:20000 (8C3) respectively detected by indirect ELISA. The antibody secreted by the two strains hybridoma cells could specifically recognized rhIL-31 detected by Western Blot, and the Antibody Subclasses was IgG3 detected by indirect ELISA. Conclusion: Express high-level recombinant human IL-31 for the IL-31 PcAb with high titer by immunizing health rabbit. (2) IL-31 PcAb decreases significantly the amount of skin inflammatory cells and Serum levels of inflammatory cytokines. Prompt IL-31 related to pathogenesis of atopic dermatitis and other skin inflammation. IL-31 PcAb has antagonistic effects. This research provides the basis for the therapeutic effects of it on dermatitis. (3)Got two strains hybridoma cells secreting strong specificity, high titer monoclonal antibody, and made a good foundation for the following research on the detection of IL-31, the biological activity and the antibody drug of IL-31.