title: [Construction and expression of single chain variable fragments (ScFv) against human CD19 antigen].
abstract: The genes encoding for the light and heavy chain variable regions were cloned by RT-PCR from a murine monoclonal hybridoma cell line, which could produce monoclonal antibody to recognize CD19 antigen on human B lymphocyte. Then fused the light and heavy chain variable regions together by a short peptide linker containing 15 amino acid (Gly4Ser)3 using splice-overlap extensive PCR. The recombinant anti-CD19- ScFv was subcloned into the expression vector pET28a and induced to be expressed by IPTG in E. coli BL21. SDS-PAGE and Western blot analysis showed that the recombinant anti-CD19-ScFv gene was expressed in E. coli BL21. ScFv expression was in the form of an inclusion bodies and the purified fusion protein was obtained after a series of purification steps including cell break, inclusion body solubilization, Ni2+ metal affinity chromatography and protein refolding. Flow cytometry analysis showed that the ScFv can react with human CD19 antigen. In conclusion, recombinant anti-CD19-ScFv gene has been successful constructed and expressed in E. coli BL21, which could provide a basic study for the future target therapy to the B lymphoid leukemia and B lymphoma.
authors.affiliation: Institute of Hematology and Blood Diseases Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin 300020, China.
authors.forename: Sen
authors.initials: S
authors.lastname: Chen
authors.forename: Qing
authors.initials: Q
authors.lastname: Rao
authors.forename: Jian-Xiang
authors.initials: JX
authors.lastname: Wang
authors.forename: Min
authors.initials: M
authors.lastname: Wang
mesh_terms: D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000235:genetics; D018941:Antigens, CD19 / Q000276:immunology*; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007128:Immunoglobulin Fragments / Q000096:biosynthesis / Q000235:genetics* / Q000276:immunology; D007135:Immunoglobulin Variable Region / Q000096:biosynthesis / Q000235:genetics* / Q000276:immunology; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics
chemical_list: D000911:Antibodies, Monoclonal; D018941:Antigens, CD19; D007128:Immunoglobulin Fragments; D007135:Immunoglobulin Variable Region; D011994:Recombinant Proteins; C055966:immunoglobulin Fv