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Generation of a mouse scFv library specific for porcine aminopeptidase N using the T7 phage display system.

Abstract
Porcine aminopeptidase N (pAPN) is a common cellular receptor for swine transmissible gastroenteritis virus (TGEV) and porcine epidemic diarrhea virus (PEDV). To investigate single-chain fragment variable (scFv) repertoire against pAPN, the genes encoding the immunoglobulin light chain variable region (VL) and heavy chain variable region (VH) were amplified by reverse transcript polymerase chain reaction (RT-PCR) using a series of degenerate primers from the spleen of BABL/c mice immunized with native pAPN. The VL and VH amplicons were combined randomly by a 12 amino acid flexible linker by splicing by overlap extension PCR (SOE-PCR), which produced the scFv gene repertoire. After ligation of the scFv gene repertoire into the T7Select10-3b vector, a mouse scFv phage library specific for pAPN was produced through in vitro packaging. The primary scFv library against pAPN contained 2.0×10(7) recombinant phage clones, and the titer of the amplified library was 3.6×10(9)pfu/mL. BstNI restriction analysis and DNA sequencing revealed that 28 phage clones from the primary pAPN scFv library showed excellent diversity. The effectiveness of the scFv library against pAPN was verified further by phage ELISA using the recombinant protein of the pAPN C subunit as coating antigen. The construction and evaluation of a murine scFv library against the common receptor pAPN of porcine coronaviruses TGEV and PEDV using the T7 phage display system are described.
AuthorsDongbo Sun, Hongyan Shi, Jianfei Chen, Da Shi, Qinghe Zhu, Hong Zhang, Shengwang Liu, Yunfeng Wang, Huaji Qiu, Li Feng
JournalJournal of virological methods (J Virol Methods) Vol. 182 Issue 1-2 Pg. 99-103 (Jun 2012) ISSN: 1879-0984 [Electronic] Netherlands
PMID22481024 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
CopyrightCopyright © 2012 Elsevier B.V. All rights reserved.
Chemical References
  • Immunoglobulin Variable Region
  • Receptors, Virus
  • Recombinant Fusion Proteins
  • Single-Chain Antibodies
  • Antigens, CD13
Topics
  • Animals
  • Antigens, CD13 (immunology)
  • Bacteriophage T7 (genetics)
  • Enzyme-Linked Immunosorbent Assay
  • Female
  • Gene Library
  • Immunoglobulin Variable Region (genetics)
  • Mice
  • Mice, Inbred BALB C
  • Porcine epidemic diarrhea virus (physiology)
  • Receptors, Virus (immunology)
  • Recombinant Fusion Proteins (genetics, immunology)
  • Restriction Mapping
  • Reverse Transcriptase Polymerase Chain Reaction
  • Sequence Analysis, DNA
  • Single-Chain Antibodies (genetics, immunology, isolation & purification)
  • Swine
  • Transmissible gastroenteritis virus (physiology)
  • Virus Attachment

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