Selection, Soluble Expression And Identification Of Full Human ScFvs Against IL-13

bjective: Since the 1980 s,with mature of the recombinant DNA technology,we widely use its in molecular biology and transform genetic structure of antibodys,technology of antibody preparation developed from a polyclonal antibody,a monoclonal antibody to genetically engineered antibody.With the continuous development of preparation for various antibody technology in recent years,,genetically engineered antibody has experienced Fab fragment,scFv antibodies,fully human antibodies,which has a small molecular weight,penetrating strong,it does not bind to Fc receptors,easy characteristics of operation,become genetically engineered antibody research focus.Bronchial asthma is a complex disease caused by a combination of multiple genetic and environmental factors.there has been no successful attempt to target any specific genetic factors to treat bronchial asthma.According to the pathogenesis of asthma from a newly discovered in molecular basis of immunology,molecular pathophysiology,and targeted to research better compliance,fewer side effects,patients can improve atopic diathesis drugs.both in mouse and human,raises the possibility that inhibition of T helper(Th)-2 cytokines such as interleukin(IL)-4,IL-5,and IL-13 could be a logical approach to asthma therapy.Interleukin plays an important role in eosinophilic and non-eosinophilic asthma,in which interleukin-13(IL-13)is closely associated with asthma cytokines.IL-13 are mainly secreted byCD4+Th2 and type 2 innate lymphoid cells,and are also produced in lesser quantities by mast cells,eosinophils,basophils,CD8+Th cells,and natural killer cells.IL-13 induced B cell proliferation and differentiation and promote IgE synthesis,activation of eosinophils and prolong survival of eosinophils,increased mucus secretion,airway hyperresponsiveness(airway hyperresponsiveness,AHR)and epithelial cells fibrosis.in this study,we will use natural fully human antibody libraries to select anti-IL-13 single chain antibody with high specificity and neutralizing properties in vitro,then we will construct anti-IL-4 /IL-13 bispecific antibodyt and research allergic asthma antibody drug.Methods : Construction of IL-13 cDNA / pET101 / D-TOPO expressionvector and connections into E.coli BL21,IPTG induced expression and purification of expressed product for identification.Biotinylated protein IL-13-antigen by using phage display technology on the natural antibody was built in the early 3 rounds of enrichmengt,detected by ELISA technique selected from enriched antibody library against IL-13.BSTNI restriction enzyme digestion method for screening of scFv against IL-13 positive clones for fingerprint analysis;The fingerprints of different clones sent to sequencing.The amber mutation,then linked the corrected gene after double digestion to LZ16 vector,transferred to E.coli DH5?F,,then expression and purification of scFv against IL-13,using Dot Blot,Western blot to identify anti-IL13-scFv protein;analysis using ELISA method to verify anti-IL-13-scFv biological function,using molecular interaction analyzer anti-IL-13 protein specificity and affinity.Results:About 700 bp of IL-13 link with pET101/D-TOPOvector;Protein expression of IL-13 is around 29 kDa in size.Identification of IL-13 by SDS-PAGE,Dot Blot,Western blot is corrected;with biotinylated protein IL-13 as antigen by using phage display antibody library against the whole source 3 rounds of enrichment through ELISA testing scFv and IL-13-binding properties of 30%;The positive clones obtained with IL-4,IL-33 by ELISA assay specific;BSTNI restriction enzyme digestion method for screening of scFv against IL-13 positive clones for fingerprint analysis;The fingerprints of different clones sent to sequencing.The amber mutation,correct sequence gene would link with LZ16;then identificated the expression,purification protein of anti-IL-13-scFv by SDS-PAGE,Dot Blot,Western blot is corrected;SDS-PAGE display the protein size of anti-IL-13-scFv at 26 kDa around.Competitive ELISA results showed: IL-13R?1 and screening single chain antibody competitive binding of IL-13,indicating that IL-13R?1 and single chain antibodies act on the same epitope of IL-13,can effectively blocking IL-13 / IL-13R?1 having the biological function of signaling pathways.Macromolecular interaction experiments(Blitz)showed that the anti-IL-13 fully human single-chain antibody and the corresponding antigen IL-13 and the resulting reaction is preferably affinity KD values 10-7,visible screened anti-IL-13 fully human single chain antibody has a good affinity.Conclusion: the specific sc Fvs against IL-13 with better affinity and neutralization had been selected successfully.