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.

Fig. 1

Amplification of the VL and VH genes from immunized mice. Lane M: DNA Marker DL2000; Lane 1: PCR products of the VH gene; Lane 2: PCR products of the VL gene; Lane 3: Amplified products of the scFv gene by SOE-PCR.

Fig. 2

Fingerprint analysis of the scFv phage clones by the frequent-cutting enzyme BstNI. Lane 1–25: The restriction patterns of each scFv clone; Lane M: DNA Marker DL2000.

Fig. 3

Phage ELISA of the phage clones generated from the third round of biopanning.