Expression, purification and activity analysis of anti-human transferrin receptor scFv.
- Author:
Li-Xia ZHAO
1
;
Bing YAN
;
Long XU
;
Shi-Wei JIANG
;
Ying-Ying ZHANG
;
Zhi-Xin YANG
;
Xiao-Wei ZHOU
;
Pei-Tang HUANG
Author Information
1. Beijing Institute of Biotechnology, Beijing 100071, China.
- Publication Type:Journal Article
- MeSH:
Animals;
Antibodies, Anti-Idiotypic;
genetics;
isolation & purification;
metabolism;
Escherichia coli;
genetics;
metabolism;
HeLa Cells;
Humans;
Immunoglobulin Fragments;
biosynthesis;
genetics;
immunology;
Immunoglobulin Variable Region;
biosynthesis;
genetics;
immunology;
Mice;
Receptors, Transferrin;
genetics;
immunology;
Recombinant Proteins;
biosynthesis;
genetics;
Transferrin;
metabolism
- From:
Chinese Journal of Biotechnology
2006;22(3):488-491
- CountryChina
- Language:Chinese
-
Abstract:
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.
