Study And Characterization Of Human G-CSF Specific ScFv Monoclonal Antibody Based On Construction A Large Human Na(?)ve Phage Display Antibody Library

1. Construction and identification of phage antibody library against rhG-CSFObjective To construct phage antibody against rhG-CSF and to develop a new way manufacturying monoclone antibody against rhG-CSF. Methods Taking human's blood cells as raw material, the phage antibody library was constructed with Kits of Recombinant Phage Antibody System and testified. Results A phage antibody library 6.42x1010cfu/ml has been constructed. Conclusion The construction of phage antibody library provides a basis for further screening and identifying it. [Key WordJ: rhG-CSF; Phage antibody library; Construction; Identification2. Screening and Identification of Phage Antibodies from Phage Display Library Against rhG-CSFObjective To obtain single chain variable fragment (ScFv)against rhG-CSF. Methods rhG-CSF was used in the panning of phage library against rhG-CSF. The activity of rhG-CSF-binding phage clone was assayed by ELISA. The specificity of expression products of the positive clones was analyzed by ELISA,SDS-PAGE and Western blotting. Results Seventy-two randomly selected clones were tested for the presence of anti-rhG-CSF ScFvs, 6 clones showed positive. The specificity of these 6 clones was confirmed by binding them to antigens of other four antigens. Two clones (G9,G32) were found to bind to rhG-CSF but not to any of the antigens of other four antigens and their expression products were about 31 kDa in size. Conclusion ScFv antibodies against the circulating antigens from rhG-CSF can be selected and manufactured from the antibody library. [Key words] rhG-CSF, phage display antibody library, screening, dentification3.Gene and Secondary Structure Analysis of Phage Antibodies Against rhG-CSFObjective To analyze genes of single-chain variable fragment(Scfv) against rhG-CSF. Methods DNA sequences of 2 positive antibody clones were determined. The software of NCBI and Ig Blast Analysis of Immunoglobulin(Ig) sequences were used to analyze their DNA sequences and deduced amino acid sequences. Secondary structures of 2 positive clones's ScFv were also modeled. Results The cloned genes of G9 and G32 were 708bp,732bp respectively and highly homologous to the heavy chain and light chain variable region's genes of human Ig. Two amino acid sequences deduced had typical features of Ig heavy and light chains' variable regions. The secondary structures were modeled with analysis software, and it was found that the sheet structure was formed by parallel peptide chain to containP-sheet and turn in the amino acid sequences of two clones. There consist with the secondary structure of Ig. Conclusion The inserts of G9 and G32 positive clones belonged to variable region's gene of human Ig.