Analysis And Identification Of Full Human ScFV Against TSLP

Objective:Antibody for the corresponding Antigen has the characteristics of high affinity and high specificity to make antibody in the diagnosis and treatment of disease has shown incomparable advantages of other types of drugs. Antibody drug has undergone monoclonal antibodies, Polyclonal Antibodies, recombinant antibody in three stages. Polyclonal Antibody from mixture b cells produce antibodies, relatively high affinity but the specificity is bad; comes from a single b cell monoclonal antibody, but much higher specificity and purity for murine antibodies and rejection for the human body; Gene engineered antibodies, also known as recombinant antibodies using recombinant DNA and protein engineering on the genes encoding antibodies prepared according to different needs renovation and the reload of antibodies, antibody currently mainly comprising a chimeric monoclonal antibody, humanized antibodies, fully human antibodies and single chain antibody, antibody drug development towards low antigen, specificity. Screened HES antibodies using in vitro filter technology became the main antibody drug research. Screened full human antibodies using in vitro filter technology became the main antibody drug research. Human thymic stromal lymphopoietin (thymic stromal lymphopoietin,TSLP) is a new, and interleukin-7 (IL-7) similar to the cytokine, when allergic inflammation in skin keratinocytes and expression in airway epithelial cells. To strongly stimulate the myeloid dendritic cells (Den-driticcell,DC) of Th2 type mediated inflammation. In genome-related research, has found repeatedly TSLP gene loci associated with susceptibility to allergic diseases. Experimental model in mice and primates confirmed to have induced TSLP is key initiator of Th2 type allergic inflammation. Study of phage antibody library by phage display technology from natural filter, full human scFv against TSLP, and characteristic analysis of scFv to filtering, provide new therapeutic approaches for allergic inflammatory diseases. Method:Amplification of TSLP cDNA, gene expression vector pET101/D-TOPO and connections into Escherichia coli BL21, IPTG induced expression and purification of expressed product for identification. Biotinylated protein TSLP-Antigen by using phage display technology on the natural antibody library was built in the early 3 rounds of enrichment, detected by ELISA technique selected from enriched single-chain antibody library full human single chain antibody (scFv) against TSLP. BSTNI restriction enzyme digestion method for screening of scFv against TSLP positive clones for fingerprint analysis; to fingerprint clones LZ16 carrier into expression and purification of the Escherichia coli BL21, Dot blot, Western blot method to identify protein expression and purification of scFv against TSLP; analysis using indirect competitive ELISA titer of scFv against TSLP protein after purification, using ELISA method to verify scFv against TSLP block function using molecular interaction Analyzer against TSLP holistic analysis of single chain antibody specificity and affinity. Results:Amplification of TSLP about 423 bp cDNA fragment size; Protein expression of TSLP is around 26 KDa in size, Dot blot, Western blot expressed protein identification displayed correctly, for the purposes of TSLP protein; with biotinylated protein TSLP as Antigen by using phage display antibody library against the whole source 3 rounds of enrichment through ELISA testing scFv and TSLP-binding properties of 35%; Will and TSLP combines capacity strong of single chain antibody for expression and the purification size for 27 KDa around; Dot blot, and Western blot identification expression purification protein and measuring sequence, results displayed right expression full human scFv against TSLP and sequence right; In non-competitive ELISA test, along with decreasing concentration of single-chain antibody protein, Antigen-binding capacity reduced, minimum single chain antibody can be diluted 100 times; molecular mutual role experiment in the full human scFv against TSLP and other cell factor no cross reaction, and corresponding Antigen TSLP reaction better of and get affinity KD value for 10"’, Visible full human scFv against TSLP with high specificity and affinity; Blocking experiments with TSLPR concentration reduced competition combined with TSLP-enhanced scFv against TSLP, scFv against TSLP to effectively block the TSLP-TSLPR signaling pathway has a biological function. Conclusion:Successfully screened to have a high specificity and affinity and biological function of full human scFv against TSLP.