Anti-CD1a Single-Chain Fv Synthesis Using Phage Display and Flow Cytometry Screening / 대한소아혈액종양학회지

PURPOSE: CD1a antigen is observed in diseases such as Langerhans cell histiocytosis, T cell acute lymphoblastic leukemia, acute myelogenous leukemia and CD1a presenting cells would be associated with transplantation disorders. In this study, anti-CD1a single-chain Fv (scFv) was made and its DNA sequence was obtained to use as useful implement and informations for the diagnosis, therapy, and study of the diseases mentioned above. METHODS: The cDNA of anti-CD1a scFv was constructed with multiple steps of PCR from NA1/34.HLK and it was inserted into pCANTAB 5 E phagemid. The anti-CD1a scFvs were expressed on transformed DH5alpha Eschericia coli and secreted into cultured media. Thymocytes were used as CD1a antigen presenting cells. ScFvs were tested with flow cytometry. DNA sequence was obtained with PCR generated DNA sequencing method. RESULTS: There were 7 clones that secreted scFvs binding thymocytes. When using thymocytes after being incubated with anti-CD1a antibody, there was only one clone (D-24) scFv of which binding reaction was significantly inhibited. DNA sequence of scFv of D-24 was obtained. CONCLUSION: The obtained scFv of D-24 should be anti-CD1a scFv because of its binding thymocytes and being inhibited by anti-CD1a antibody. It needs further purification and confirmative test such as immunoprecipitation. The obtained DNA sequence would be informative to construct various and humanized antibodies for CD1a antigen.

Production and expression of a single chain antibody against platelet in E. coli / 中国免疫学杂志

Objective:To express the recombinant single chain Fv(scFv) in E.coli and reduce immunogenicity and molecular weight of a monoclonal antibody specific for human platelet.Methods:The variable regions of the heavy and light chains of platelet specific antibody SZ 2 were amplified by reverse transcription and polymerase chain reaction.VH and VL gene segments were cloned into pUC Tm and joined together with a (gly 4ser) 3 linker.The resulting scFv was expressed in PET expression system.The expressed recombinant protein was characterized by its size on SDS PAGE,by Western blot,by flow cytometry and its functions.Results:The VH and VL genes were homologous with the published gene sequences of mouse antibody variable region.The recombinant scFv was expressed mostly in the form of inclusion bodies,and the yield was up to 25% of the total cell proteins.Functional studies showed that SZ 2 scFv could bind to platelet and could suppress platelet aggregation induced by ristocatin and thrombin.Conclusion:A recombinant SZ 2 scFv specific against platelet was developed and characterized.

Establishment of large phage antibody library / 中国免疫学杂志

Objective:To create a large human phage antibody library from which easy to get diabody and select human antibody clones.Methods:VL and VH genes were amplified by RT-PCR from RNA which came from normal adult peripheral blood and new-born cord blood lymphocytes. VL and VH genes were reamplified to add a region of overlap in the ScFv linker to form ScFv genes in which VH genes were flanked by two non-homologous loxp sites. The ScFv genes were cloned into PDF to obtain a primary library. This primary library was used to infect bacteria Bs1365(expressing cre recombinase) with high multiplicity of infection(MOI) 200∶1. This procedure resulted in a very large phage antibody library through VL and VH recombined by the cre recombinase. Following recombination, phagemide were derived from these bacteria and used to infect bacteria not expressing cre(XL1-Blue) at MOI