[Construction, expression and functional characterization of single chain variable fragments (scFv) against human CD33 antigen].
OBJECTIVE
To construct and express the single chain variable fragments (scFv) gene against human CD33 antigen, and characterize its bioactivity.


METHODS
The genes encoding the light and heavy chain variable regions were cloned by RT-PCR from a murine hybridoma cell line, which could produce monoclonal antibody(mAb) against human CD33 antigen. Then the light and heavy chain variable regions were fused together by a short peptide linker containing 15 amino acid (Gly(4)Ser)(3) using splice-overlap extensive PCR. The recombinant anti-CD33 scFv was subcloned into the expression vector pET28a(+) and expressed in E.coli Rosetta after induction by IPTG.


RESULTS
SDS-PAGE and Western blot analysis showed that the recombinant anti-CD33 scFv gene was expressed in the form of inclusion body in E.coli Rosetta, and the purified fusion protein was obtained after a series of purification steps including cell lysis, inclusion body solubilization, Ni(2+) metal affinity chromatography and protein refolding. Flow cytometry(FCM) analysis showed that the scFv could react with human CD33 antigen.


CONCLUSIONS
Recombinant anti-CD33 scFv gene has been successfully constructed and expressed in E.coli Rosetta, which could provide foundation for the future target therapy to the myeloid leukemia.
Institute of Hematology and Blood Diseases Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin 300020, China. aiguozhe2001@163.com
Xiao-Jun
XJ
Chen
Yang
Y
Wang
Hao
H
Qu
Xin-Shun
XS
Ge
Yu-Feng
YF
Zuo
Xiao-Long
XL
Liao
D000818:Animals; D015703:Antigens, CD / Q000276:immunology*; D015214:Antigens, Differentiation, Myelomonocytic / Q000276:immunology*; D001667:Binding, Competitive; D015153:Blotting, Western; D045744:Cell Line, Tumor; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D005455:Fluorescent Antibody Technique; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D007143:Immunoglobulin Heavy Chains / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D007147:Immunoglobulin Light Chains / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D007135:Immunoglobulin Variable Region / Q000096:biosynthesis / Q000235:genetics* / Q000276:immunology* / Q000302:isolation & purification; D063268:Sialic Acid Binding Ig-like Lectin 3
D015703:Antigens, CD; D015214:Antigens, Differentiation, Myelomonocytic; C571163:CD33 protein, human; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D007135:Immunoglobulin Variable Region; D063268:Sialic Acid Binding Ig-like Lectin 3