Homology modelling and bivalent single-chain Fv construction of anti-HepG2 single-chain immunoglobulin Fv fragments from a phage display library

Homology modelling and BsFv construction of anti-HepG2 scFv 691

MING NI3, BING YU*1, YU HUANG2, ZHENJIE TANG2, PING LEI2, XIN SHEN2, WEI XIN2,

HUIFEN ZHU2 and GUANXIN SHEN21Department of Pathogen Biology, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China

2Department of Immunology, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China

3Department of Infectious Diseases, Tongji Hospital, Tongji Medical College, Huazhong University of Science and

Technology, Wuhan 430030, China

Corresponding author (Email, [email protected])

We prepared single-chain immunoglobulin Fv fragments (scFv) SLH10 specic for the HepG2 cell line after biopanning from a large human-nave phage display library (Grifn. 1 Library). The three-dimensional (3D) structure of SLH10 was modelled by the Insight II molecule simulation software. The structure was rened using the molecular dynamics method. The structures with the least steric clashes and lowest energy were determined nally. The optimized structures of heavy (VH) and light (VL) variable chains of SLH10 scFv were obtained. Then SLH10 bivalent single-chain Fv (BsFv) was constructed that would be suitable for high-afnity targeting. SLH10 BsFv was generated by linking scFvs together and identied by sequencing. Its expression products were conrmed by western blot analysis. The relative molecular masses of scFv and BsFv were approximately 30 kDa and 60 kDa, respectively. Flow cytometry revealed that SLH10 BsFv bound the selected cell lines with greater signal intensity than the parental scFv. The improved antigen binding of SLH10 BsFv may be useful for immunodiagnostics or targeted gene therapy for liver cancer.

[Ni M, Yu B, Huang Y, Tang Z, Lei P, Shen X, Xin W, Zhu H and Shen G 2008 Homology modelling and bivalent single-chain Fv construction of anti-HepG2 single-chain immunoglobulin Fv fragments from a phage display library; J. Biosci. 33 691697]

1. Introduction

Single-chain immunoglobulin (Ig) Fv fragments (scFvs) are predominantly monomeric when the linker variable regions of the heavy (VH) and light (VL) chains are joined by polypeptide linkers (Adair 1992). scFvs maintain the binding specicity of the parental antibody; they are in

Keywords. BsFv; homology modelling; scFv; three-dimensional (3D) structure

Abbreviations used: AMBER, assisted model building and energy; BSA, bovine serum albumin; BsFv, bivalent single-chain Fv; CDR, complementary determining region; CVFF, consistent valence force eld; 3D, three-dimensional; FBS, foetal bovine serum; FITC, uorescein...