Summary
To obtain single chain variable fragment (scFv) and bivalent single chain variable fragment (bsFv) against transferrin receptor, up-stream and down-stream primers were designed according to the complementary sequences of FR1 region of variable heavy (VH) and FR4 of variable light (VL), respectively, which contained inter-linker G4S and the restriction endonuclease SfiI, AscI and NotI. Two pieces of scFv fragments were first amplified through PCR and then inserted into plasmid pAB1, which could express scFv protein once induced by IPTG in the host bacteria. To express scFv and bsFv, E. coli TG1 was cultured in LB broth and was induced by IPTG. The restriction enzyme digestion map and DNA sequencing demonstrated that scFv and bsFv genes were successfully inserted into the expression plasmid. SDS-PAGE and Western blotting revealed the protein band at 35kD and 60kD, which were consistent with the molecular weight of scFv and bsFv respectively. Flow cytometry showed that scFv and bsFv harbored the specific binding activity with TfR expressed in various tumor cells, and the avidity of bsFv was higher than that of the parent scFv.
Similar content being viewed by others
References
Daniels T R, Delgado T, Rodriguez J A et al. The transferrin receptor part I: biology and targeting with cytotoxic antibodies for the treatment of cancer. Clin Immunol, 2006,121(2):144–158
Qing Y, Shuo W, Zhihua W et al. The in vitro antitumor effect and in vivo tumor-specificity distribution of human-mouse chimeric antibody against transferrin receptor. Cancer Immunol Immunother, 2006,55(9):1111–1121
Lei P, He Y, Ye Q et al. Antigen-binding characteristics of AbCD71 and its inhibitory effect on PHA-induced lymphoproliferation. Acta Pharmacol Sin, 2007,28(10): 1659–1664
Bièche I, Olivi M, Champème M H et al. Novel approach to quantitative polymerase chain reaction using real time detection:application to the detection of gene amplification in breast cancer. Int J Cancer, 1998,78(5):661–666
Kohgo Y, Kondo H, Mogi Y et al. Mechanism and clinical significance of soluble hepatic cell-surface receptors. Targeted Diagn Ther, 1991,4:305–319
Widera A, Norouziyan F, Shen W C. Mechanisms of TfR-mediated transcytosis and sorting in epithelial cells and applications toward drug delivery. Adv Drug Deliv Rev, 2003,55(11):1439–1466
Kollia P, Samara M, Stamatopoulos K et al. Molecular evidence for transferrin receptor 2 expression in all FAB subtypes of acutemyeloid leukemia. Leuk Res, 2003,27(12):1101–1103
Yang D C, Wang F, Elliott R L et al. Expression of transferrin receptor and ferritin H-chain mRNA are associated with clinical and histopathological prognostic indicators in breast cancer. Anticancer Res, 2001,21(1B):541–549
Takubo T, Kumura T, Nakao T et al. Clinical usefulness of combined measurements of serum soluble transferrin receptor levels and serum interleukin-18 levels at determination of serum KL-6 levels in haematologic malignancies. Acta Haematol, 2000,104(2–3):141–143
Daniels T R, Delgado T, Helguera G et al. The transferrin receptor part II: targeted delivery of therapeutic agents into cancer cells. Clin Immunol, 2006,121(2):159–176
Pirollo K F, Dagata J, Wang P et al. A tumor-targeted nanodelivery system to improve early MRI detection of cancer. Mol Imaging, 2006,5(1):41–52
Asai T, Trinh R, Penichet M L et al. A human bioti acceptor domain allows site-specific conjugation of an enzyme to an antibody-avidin fusion protein for targeted drug delivery. Biomol Eng, 2005,21(6):145–155
Rodríguez J A, Helguera G, Daniels T R. Binding specificity and internalization properties of an antibody-avidin fusion protein targeting the human transferrin receptor. J Control Release, 2007,124(1–2):35–42
Hsu C P, Ko J L, Shai S E et al. Modulation of telomere shelterin by TFR1 and TRF2 interacts with telomerase to maintain the telomere length in non-small cell lung cancer. Lung Cancer, 2007,58(3):310–316
Högemann-Savellano D, Bos E, Blondet C et al. The transferrin receptor: a potential molecular imaging marker for human cancer. Neoplasia, 2003,5(6):495–506
Shinohara H, Fan D, Ozawa S et al. Site-specific expression of transferrin receptor by human colon cancer cells directly correlates with eradication by an titransferrin recombinant immunotoxin. Int J Oncol, 2000,17(4): 643–651
Bhatia J, Sharma S K, Chester K A et al. Catalytic activit of an in vivo tumor targeted anti-CEA scFv: carboxypeptidase G2 fusion protein. Int J Cancer, 2000,85(4):571–576
Kuroki M, Arakawa F, Khare P D et a1. Specific targeting strategies of cancer gene therapy using a single-chain variable fragment (scFv) with a high affinity for CEA. Anticancer Res, 2000,20(6A):4067–4071
Yang D F, Zhu H F, Shen G X et al. Construction of single chain fv antibody against transferrin receptor and its protein fusion with alkaline phosphatase. World J Gastroenterol, 2005,11(21):3300–3304
Peng J L, Wu S, Zhao X P et al. Downregulation of transferrin receptor surface expression by intracellular antibody. Biochem Bioph Res Co, 2007,354(4):864–871
Author information
Authors and Affiliations
Additional information
Jing LIU, female, born in 1981, Doctorial Candidate
This project was supported by a grant from “863” program of China (No. 2006AA02Z158), the Ministry of Education Science Foundation of China (No. 20060487024) and Science and Technology project of Jiangxi Province Education Department (No. 2006-86).
Rights and permissions
About this article
Cite this article
Liu, J., Xiao, D., Zhou, X. et al. Preparation and identification of scFv and bsFv against transferrin receptor. J. Huazhong Univ. Sci. Technol. [Med. Sci.] 28, 621–625 (2008). https://doi.org/10.1007/s11596-008-0601-z
Received:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s11596-008-0601-z