Generation of a mouse scFv library specific for porcine aminopeptidase N using the T7 phage display system.
Generation of a mouse scFv library specific for porcine aminopeptidase N using the T7 phage display system.
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使用 T7 噬菌体展示系统生成猪氨肽酶 N 特异性的小鼠 scFv 文库
DOI：
10.1016/j.jviromet.2012.03.021
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发表时间：
2012-06
影响因子：
3.1
通讯作者：
Feng L
中科院分区：
文献类型：
--
作者：
Sun D;Shi H;Chen J;Shi D;Zhu Q;Zhang H;Liu S;Wang Y;Qiu H;Feng L
利用T7 Select噬菌体展示技术构建抗pAPN的单链抗体库。scFv文库具有足够大的容量。►单链抗体库具有足够的多样性。猪氨肽酶N（pAPN）是猪传染性胃肠炎病毒（TGEV）和猪流行性腹泻病毒（PEDV）共同的细胞受体。为了研究针对pAPN的单链可变区（single chain fragment variable，scFv）库，使用一系列简并引物，通过逆转录聚合酶链反应（reverse transcriptpolymerase chain reaction，RT-PCR）从天然pAPN免疫的BABL/c小鼠脾脏中扩增编码免疫球蛋白轻链可变区（light chain variable region，VL）和重链可变区（heavy chain variable region，VH）的基因。VL和VH扩增子通过重叠延伸PCR剪接（SOE-PCR）通过12个氨基酸的柔性接头随机组合，其产生scFv基因库。将scFv基因库连接到T7 Select 10 -3b载体后，通过体外包装产生针对pAPN的小鼠scFv噬菌体文库。初步构建的抗pAPN单链抗体库含有2.0 × 107个重组噬菌体克隆，扩增后的抗体滴度为3.6 × 109 pfu/mL。BstNI酶切分析和DNA测序结果表明，pAPN单链抗体库中的28个噬菌体克隆具有良好的多样性。以pAPN C亚基的重组蛋白为包被抗原，通过噬菌体ELISA进一步验证了抗pAPN单链抗体库的有效性。利用噬菌体T7展示系统构建了抗猪冠状病毒TGEV和PEDV共同受体pAPN的单链抗体库。
► A simple and rapid measure for generation of a scFv library against pAPN using T7Select phage display. ► The scFv library possesses a sufficiently large capacity. ► The scFv library has a sufficient diversity. 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 × 107 recombinant phage clones, and the titer of the amplified library was 3.6 × 109 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.
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影响因子：
2.7
作者：
Reynolds DJ;Garwes DJ
通讯作者：
Garwes DJ
猪氨肽酶 N 是 PEDV 冠状病毒的功能受体
影响因子：
3.7
作者：
Li, B. X.;Ge, J. W.;Li, Y. J.
通讯作者：
Li, Y. J.
猪氨肽酶N在原核表达系统中的表达及功能分析
影响因子：
4.1
作者：
Liu B;Li G;Sui X;Yin J;Wang H;Ren X
通讯作者：
Ren X
影响因子：
3.7
作者：
Ribes JM;Ortego J;Ceriani J;Montava R;Enjuanes L;Buesa J
通讯作者：
Buesa J
影响因子：
3.1
作者：
Elia G;Decaro N;Martella V;Lorusso E;Mari V;Maria SL;Cordioli P;Buonavoglia C
通讯作者：
Buonavoglia C