[Construction and expression of anti-human integrin alphavbeta3 scFv].
OBJECTIVE
To construct single chain antibody (scFv) gene of mAb E10 against human integrin alphavbeta3.


METHODS
The VH and VL genes were amplified from hybridoma cells secreting mAb E10 by RT-PCR and connected with the use of linker (Gly4Ser)3 to assemble scFv gene. The scFv gene was cloned into prokaryotic expression vector pTIG-TRX and expressed in E. coli BL21 (DE3).


RESULTS
SDS-PAGE analysis showed the expressed recombinant protein with relative molecular mass (Mr) being 31,000. Western blot confirmed that the protein was labeled with His6. scFv protein was expressed as soluble protein under the condition of a small amount of IPTG induction and culture at lower temperature. The purity of the protein purified through Ni-NTA agarose metal affinity resin column was over 91%. The purified protein could bind to the human integrin alphavbeta3 by ELISA confirmation.


CONCLUSIONS
scFv against human integrin alphavbeta3 has been successfully constructed and expressed,which lays the foundation for further clinical research.
Wang
Chen
C
Institute of Microbiology & Epidemiology, Academy of Military Medical Sciences, Beijing 100071, China. wangchen2001@163.com
Hou
Li-hua
LH
Zhang
Yan-ming
YM
Li
Jian-min
JM
Liao
Zhen-lin
ZL
Du
Gui-xin
GX
Chen
Wei
W
Sun
Qi-hong
QH
Tong
Yi-gang
YG
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000235:genetics; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D004926:Escherichia coli / Q000378:metabolism; D050438:Genes, Immunoglobulin Heavy Chain; D050439:Genes, Immunoglobulin Light Chain; D005822:Genetic Vectors; D006801:Humans; D006825:Hybridomas / Q000378:metabolism; D007128:Immunoglobulin Fragments / Q000096:biosynthesis* / Q000235:genetics; D039302:Integrin alphaVbeta3 / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D051379:Mice; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics
D000911:Antibodies, Monoclonal; D018076:DNA, Complementary; D007128:Immunoglobulin Fragments; D039302:Integrin alphaVbeta3; D011994:Recombinant Proteins; C055966:immunoglobulin Fv