Recombinant Expression And Production Of Monoclonal Antibody Against Interleukin-9Protein

Interleukin-9, which was named P40(IL-9).was first described from a multiple ofestablished Th2cell lines at1988by Uyttenhove[1].Then IL-9and IL-10was secreted by anew Th9cell which was reported by Nature Immunology publication in2008. Thescientific community has entered a new upsurge in the study of IL-9.As reported in the literature, IL-9was related with allergic asthma, parasitic infection,and genetic allergies disease. Asthma disease leads to the high expression of the IL-9. Then,IL-9activate mast cell and eosinophils. As a result, airway inflammation are increased withhigh anaphylaxis[2]. In the parasitic infection, IL-9is involved in the immune protectiveeffect[3]. These reports showed that the IL-9monoclonal antibody with the popularmodification could effectively block the expression of IL-9in vivo, and thus play a role inmitigation on the treatment of asthma disease. And this would lay the foundation for themonoclonal antibody drug research&development in the future.In this study, the gene of IL-9in the GeneBank mature peptide, whose codon wasreplaced according to the E. coli codon biased tropism, was synthesized by Shanghai jerrybio Co.,Ltd, and cloned into the vector PET-22b.The IL-9gene was amplified by PCR, when the PET-22b-IL-9plasmid was used as atemplate. And the gene and the pGEX-6P-2vector connection was transferred into theXL-1Blue (E. coli). The best expression conditions tested were:30℃0.2mmol/L IPTG,induction for5hours. SDS-PAGE electrophoresis analysis showed that the expression ofthe target protein were both in the supernatant and inclusion body. Purified by GST affinitychromatography, high purity of recombinant IL-9protein was obtained.BALB/C mice were immunized with the purified protein, and anti-serum for1:16×104titer were obtained. The spleen cells from immunized mice were hybridized with SP2/0cells using PEG-1500. The hybridization was at42%. Indirect ELISA results showed thatthe positive rate of monoclonal in the supernatant of the successful fusion cell was at3.6%.After three times sub-cloning and repetitive selection, one hybrid cell line was acquirednamed2E5. Colchicine identification hybridoma results showed that the cell lines wasbelonged to the integration of the cell body. Specific experiment demonstrated that the2E5antibody which possessed high specificity could specifically react with recombinant IL-9,standard protein IL-9and GST fusion protein. Indirect ELISA results showed that thishybrid cell line could steadily secrete monoclonal antibody after repeated thawing andfreezing, the hybridoma cell supernatant and ascites antibody titer maintained at1:320,1:12800respectively. In summary, the success of this hybridoma laid the foundationfor the antibody drug development.