[Construction and expression of mouse-human chimeric Fab fragment gene of monoclonal antibody SZ-2 against platelet].
OBJECTIVE
To reduce immunogenicity of a monoclonal antibody SZ-2 specific for human platelet.


METHODS
Reverse transcription and polymerize chain reaction were used to amplify the variable region genes of monoclonal antibody SZ-2. The cloned V(H) and V(L) genes were sequenced and fused to human IgG1 constant region gene CH1 and Ckappa in plasmid pSW1. The recombinant plasmid were transformed into E. coli. The expressed recombinant proteins were analysed.


RESULTS
The V(H) and V(L) genes were homologous with the published gene sequences of mouse antibody variable region. The concentration of chimeric Fab fragment in expression supernatant was about 180 microg/L detected by ELISA. Western blot analysis showed that SZ-2 Fab/Hu maintained the binding activity to human platelet GPIb. The recombinant proteins could suppress platelet aggregation induced by Ristocatin.


CONCLUSIONS
The variable region genes of SZ-2 are cloned and the mouse-human chimeric Fab fragment is expressed successfully in E. coli.
Dai
Ke-sheng
KS
The First Affiliated Hospital of Suzhou University, Jiangsu Institute of Hematology, Suzhou 215006, China. uujihsmc@public1.sz.cn
Zhu
Huai-ping
HP
Jiang
Miao
M
Ruan
Chang-geng
CG
D000818:Animals; D000911:Antibodies, Monoclonal / Q000235:genetics*; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007127:Immunoglobulin Constant Regions / Q000235:genetics; D007140:Immunoglobulin Fab Fragments / Q000235:genetics*; D007074:Immunoglobulin G / Q000235:genetics*; D007143:Immunoglobulin Heavy Chains / Q000235:genetics; D007147:Immunoglobulin Light Chains / Q000235:genetics; D007135:Immunoglobulin Variable Region / Q000235:genetics; D051379:Mice; D010957:Plasmids; D010974:Platelet Aggregation / Q000187:drug effects; D019038:Platelet Glycoprotein GPIb-IX Complex / Q000276:immunology*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000494:pharmacology; D014170:Transformation, Genetic
D000911:Antibodies, Monoclonal; D007127:Immunoglobulin Constant Regions; D007140:Immunoglobulin Fab Fragments; D007074:Immunoglobulin G; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D007135:Immunoglobulin Variable Region; D019038:Platelet Glycoprotein GPIb-IX Complex; D011993:Recombinant Fusion Proteins; C023592:glycocalicin