An Tong-Qing, Zhou Yan-Jun, Qiu Hua-Ji, Tong Guang-Zhi, Wang Yun-Feng, Liu Jin-Xia, Yang Jin-Yu
National Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, CAAS, 427 Maduan Street, 150001, Harbin, People's Republic of China.
Virus Genes. 2005 Aug;31(1):81-7. doi: 10.1007/s11262-005-2203-1.
A phage display peptide library targeting the nucleocapsid (N) protein of porcine reproductive and respiratory syndrome virus (PRRSV) strain CH-1a was generated and used for epitope mapping. After 3 rounds of biopanning with the monoclonal antibody (MAb) N3H2 directed against the N protein, 3 positive phages were screened and sequenced. These phages share a consensus sequence, IQTAFNQGA, which corresponds to the amino acid (AA) 79-87 segment of the CH-1a N protein. A small DNA fragment coding for IQTAFNQGA was expressed as a fusion product, and reacted to N3H2 in Western blots and indirect ELISA. Four truncated peptides (IQTAFNQG, IQTAFNQ, QTAFNQGA, and TAFNQGA) expressed as GST fusion products failed to react with N3H2. The sequences around the N3H2-binding site among the N proteins of 57 PRRSV strains were compared. Our results indicate that the IQTAFNQGA motif is highly conserved among North American and European isolates. We concluded that the precisely defined nona-peptide epitope is a novel conserved Linear B cell epitope on the N protein of PRRSV.
构建了一个针对猪繁殖与呼吸综合征病毒（PRRSV）CH-1a株核衣壳（N）蛋白的噬菌体展示肽库，并用于表位定位。用针对N蛋白的单克隆抗体（MAb）N3H2进行3轮生物淘选后，筛选出3个阳性噬菌体并进行测序。这些噬菌体共有一个一致序列IQTAFNQGA，它对应于CH-1a N蛋白的氨基酸（AA）79 - 87片段。编码IQTAFNQGA的一个小DNA片段作为融合产物表达，并在蛋白质免疫印迹和间接酶联免疫吸附测定中与N3H2发生反应。以谷胱甘肽S-转移酶（GST）融合产物形式表达的4个截短肽（IQTAFNQG、IQTAFNQ、QTAFNQGA和TAFNQGA）未能与N3H2发生反应。比较了57株PRRSV毒株N蛋白中N3H2结合位点周围的序列。我们的结果表明，IQTAFNQGA基序在北美和欧洲分离株中高度保守。我们得出结论，精确界定的九肽表位是PRRSV N蛋白上一个新的保守线性B细胞表位。
