Selection of scFvs specific for the HepG2 cell line using ribosome display.
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.
Zhou
Lei
L
Jiangsu Province Key Laboratory of Molecular and Medical Biotechnology, College of Life Sciences, Nanjing Normal University, Nanjing 210046, P R China.
Mao
Wei-Ping
WP
Fen
Juan
J
Liu
Hong-Yun
HY
Wei
Chuan-Jing
CJ
Li
Wen-Xiu
WX
Zhou
Feng-Yun
FY
D000818:Animals; D006528:Carcinoma, Hepatocellular / Q000378:metabolism; D002460:Cell Line; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D006801:Humans; D007128:Immunoglobulin Fragments; D007143:Immunoglobulin Heavy Chains / Q000737:chemistry*; D008113:Liver Neoplasms / Q000737:chemistry / Q000378:metabolism*; D051379:Mice; D008807:Mice, Inbred BALB C; D008954:Models, Biological; D016133:Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012270:Ribosomes / Q000737:chemistry / Q000378:metabolism*
D007128:Immunoglobulin Fragments; D007143:Immunoglobulin Heavy Chains