Division of Swine Infectious Diseases, National Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute of the Chinese Academy of Agricultural Sciences, Nangang District, Harbin 150001, China.
J Virol Methods. 2012 Jun;182(1-2):99-103. doi: 10.1016/j.jviromet.2012.03.021. Epub 2012 Mar 28.
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.
猪氨基肽酶 N（pAPN）是猪传染性胃肠炎病毒（TGEV）和猪流行性腹泻病毒（PEDV）的常见细胞受体。为了研究针对 pAPN 的单链片段可变（scFv）文库，使用一系列从用天然 pAPN 免疫的 BABL/c 小鼠脾脏中扩增的简并引物，通过逆转录聚合酶链反应（RT-PCR）扩增编码免疫球蛋白轻链可变区（VL）和重链可变区（VH）的基因。VL 和 VH 扩增子通过拼接重叠延伸 PCR（SOE-PCR）随机组合由 12 个氨基酸的柔性接头，产生 scFv 基因文库。将 scFv 基因文库连接到 T7Select10-3b 载体后，通过体外包装产生了针对 pAPN 的小鼠 scFv 噬菌体文库。针对 pAPN 的初级 scFv 文库包含 2.0×10(7)个重组噬菌体克隆，扩增文库的滴度为 3.6×10(9)pfu/mL。BstNI 限制分析和 DNA 测序表明，从初级 pAPN scFv 文库中 28 个噬菌体克隆显示出极好的多样性。通过使用 pAPN C 亚基重组蛋白作为包被抗原的噬菌体 ELISA 进一步验证了 scFv 文库对 pAPN 的有效性。描述了使用 T7 噬菌体展示系统构建和评估针对猪冠状病毒 TGEV 和 PEDV 的共同受体 pAPN 的鼠 scFv 文库。
