Construction and expression of single chain variable fragments (ScFv) against human CD19 antigen.
- Author:
Sen CHEN
1
;
Qing RAO
;
Jian-Xiang WANG
;
Min WANG
Author Information
1. Institute of Hematology and Blood Diseases Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin 300020, China.
- Publication Type:Journal Article
- MeSH:
Antibodies, Monoclonal;
biosynthesis;
genetics;
Antigens, CD19;
immunology;
Cloning, Molecular;
Escherichia coli;
genetics;
metabolism;
Humans;
Immunoglobulin Fragments;
biosynthesis;
genetics;
immunology;
Immunoglobulin Variable Region;
biosynthesis;
genetics;
immunology;
Recombinant Proteins;
biosynthesis;
genetics
- From:
Chinese Journal of Biotechnology
2005;21(5):686-691
- CountryChina
- Language:Chinese
-
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.
