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Selection of scFvs specific for the HepG2 cell line using ribosome display

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Abstract

The aim of this study was to construct a ribosome display library of single chain variable fragments (scFvs) associated with hepatocarcinoma and screen such a library for hepatocarcinoma-binding scFvs. mRNA was isolated from the spleens of mice immunized with hepatocellular carcinoma cell line HepG2. Heavy and k chain genes (VH and k) were amplified separately by RT-PCR, and an anti-HepG2 VH/k chain ribosome display library was constructed by assembling VH and k into the VH/k chain with a specially constructed linker by SOE-PCR. The VH/k chain library was transcribed and translated in vitro using a rabbit reticulocyte lysate system. In order to isolate specific scFvs, recognizing HepG2 negative selection on a normal hepatocyte line WRL-68 was carried out before three rounds of positive selection on HepG2. After three rounds of panning, cell enzyme-linked immunosorbent assay (ELISA) showed that one of the scFvs had high affinity for the HepG2 cell and lower affinity for the WRL-68 cell. In this study, we successfully constructed a native ribosome display library. Such a library would prove useful for direct intact cell panning using ribosome display technology. The selected scFv had a potential value for hepatocarcinoma treatment.

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Abbreviations

ARM:

antibody-ribosome-mRNA

BSA:

bovine serum albumin

CDR:

complementarity-determining region

DMEM:

Dulbecco modified Eagle high glucose medium

ELISA:

enzyme-linked immunosorbent assay

FBS:

foetal bovine serum

IPTG:

isopropyl-b-D-thiogalactopyranoside

PBS:

phosphate buffered saline

RT-PCR:

reverse transcriptase-polymerase chain reaction

scFv:

single-chain variable fragment

SDS-PAGE:

sodium dodecyl sulphate-polyacrylamide gel electrophoresis

VH:

variable region of heavy chain

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Authors and Affiliations

  1. Jiangsu Province Key Laboratory of Molecular and Medical Biotechnology, College of Life Sciences, Nanjing Normal University, Nanjing, 210046, P R China

    Lei Zhou, Wei-Ping Mao, Juan Fen, Hong-Yun Liu, Chuan-Jing Wei, Wen-Xiu Li & Feng-Yun Zhou

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  1. Lei Zhou
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  2. Wei-Ping Mao
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  3. Juan Fen
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  4. Hong-Yun Liu
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  5. Chuan-Jing Wei
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  6. Wen-Xiu Li
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  7. Feng-Yun Zhou
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Correspondence to Wei-Ping Mao.

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Zhou, L., Mao, WP., Fen, J. et al. Selection of scFvs specific for the HepG2 cell line using ribosome display. J Biosci 34, 221–226 (2009). https://doi.org/10.1007/s12038-009-0026-2

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  • DOI: https://doi.org/10.1007/s12038-009-0026-2

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