[Study of a humanized anti-platelet GP VI single chain Fv phage antibody].
OBJECTIVE
To study the anti-platelet GPVI single chain Fv phage antibody which can inhibit the aggregation function of platelet by using phage antibody library technology.


METHODS
ITP patients with anti-platelet GPVI autoantibody that could inhibit the aggregation function of platelet were screened by MAIPA assay and platelet aggregation test. The gene fragments of heavy chain and light chain variable region (VH and VL) of immunoglobulin were amplified by RT-PCR from peripheral blood lymphocytes mRNA of the screened patients. The VH and and VL fragments were linked through a DNA linker encoding the peptide (Gly4Ser)3 to construct single chain Fv (ScFv) gene. The ScFv gene was digested with SfiI/NotI restriction enzymes and cloned into the pHEN2 phage display vector, then electrically transformed to E. coli TG1. The TG1 containing ScFv-pHEN2 was rescued by helper phage M13K07 to produce ScFv phage antibody. The anti-platelet GPVI phage ScFv antibody was enriched and purified. The effect of the phage antibody on platelet aggregation function was studied.


RESULTS
Of 806 chronic ITP patients, 11 (1.36%) were positive for anti-platelet GPVI autoantibody and 2 (0.24%) patients'plasma significantly inhibited the collagen induced platelet aggregation. The length of VH and VL fragments was about 380 to 400 bp, and were successfully formed ScFv fragments of about 800 bp by DNA linker. After cloning ScFv to phagemid vector pHEN2 and transforming ScFv-pHEN2 to TG1, 4.1x10(7) clones were obtained. After M13K07 rescue, 2.62x10(10) cfu/ml ScFv phage antibodies were produced. The purified anti-platelet GPVI ScFv phage antibody inhibited the collagen induced platelet aggregation.


CONCLUSIONS
Anti-platelet GPVI ScFv phage antibody produced by phage antibody library technology can inhibit the aggregation function of platelet.
Ji
Xue-bin
XB
Department of Hematology, the Tumor Center, QiLu Hospital of Shandong University, Jinan 250012, China.
Hou
Ming
M
Zhang
Chun-qing
CQ
Shi
Yan
Y
Peng
Jun
J
Chu
Xiao-xia
XX
Ma
Dao-xin
DX
Li
Li-zhen
LZ
D000293:Adolescent; D000328:Adult; D000368:Aged; D004926:Escherichia coli / Q000235:genetics; D005260:Female; D006801:Humans; D008297:Male; D008875:Middle Aged; D019151:Peptide Library; D010974:Platelet Aggregation / Q000276:immunology; D010975:Platelet Aggregation Inhibitors / Q000276:immunology / Q000494:pharmacology*; D010980:Platelet Membrane Glycoproteins / Q000276:immunology*; D057127:Single-Chain Antibodies / Q000235:genetics* / Q000276:immunology / Q000494:pharmacology*; D014169:Transformation, Bacterial; D055815:Young Adult
D019151:Peptide Library; D010975:Platelet Aggregation Inhibitors; D010980:Platelet Membrane Glycoproteins; D057127:Single-Chain Antibodies; C098341:platelet membrane glycoprotein VI