Selection of scFvs specific for the HepG2 cell line using ribosome display |
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Authors: | Lei Zhou Wei-Ping Mao Juan Fen Hong-Yun Liu Chuan-Jing Wei Wen-Xiu Li Feng-Yun Zhou |
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Institution: | (1) Jiangsu Province Key Laboratory of Molecular and Medical Biotechnology, College of Life Sciences, Nanjing Normal University, Nanjing, 210046, P R China |
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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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Keywords: | Hepatocarcinoma ribosome display single-chain antibody |
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