[Expression, purification and activity analysis of anti-human transferrin receptor scFv].
Human transferrin receptor (TfR) was isolated from homogenates of placental tissues by affinity chromatography on transferrin-Sepharose, and then used to screen human scFv against it from a fully-synthesized phage scFv library. After verifying the specificity, gene fragment of one of the selected scFv was inserted into the plasmid pET22b(+) and transformed into E. coli BL21(DE3) . Expression of scFv in transformant was induced with 0.5mmol/L IPTG. ELISA assay on HeLa cells showed that scFv protein could recognize and bind to TfR on the surface of HeLa cells. The scFv was purified by one-step affinity chromatography with Ni+ -NTA agarose, and injected into Kunming mouse via tail veins. This scFv was detected in brain tissues 1h later by capillary depletion method, which indicates that scFv protein can permeate through the blood brain barrier by mediation of the TfR receptor. Our works lay the foundation for the treatment of tumors and central nervous system diseases.
Zhao
Li-Xia
LX
Beijing Institute of Biotechnology, Beijing 100071, China.
Yan
Bing
B
Xu
Long
L
Jiang
Shi-Wei
SW
Zhang
Ying-Ying
YY
Yang
Zhi-Xin
ZX
Zhou
Xiao-Wei
XW
Huang
Pei-Tang
PT
D000818:Animals; D000888:Antibodies, Anti-Idiotypic / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D007128:Immunoglobulin Fragments / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D007135:Immunoglobulin Variable Region / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D051379:Mice; D011990:Receptors, Transferrin / Q000235:genetics / Q000276:immunology*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D014168:Transferrin / Q000378:metabolism
D000888:Antibodies, Anti-Idiotypic; D007128:Immunoglobulin Fragments; D007135:Immunoglobulin Variable Region; D011990:Receptors, Transferrin; D011994:Recombinant Proteins; D014168:Transferrin