Aquatic Biotechnology Center, College of Veterinary Medicine, Gyeongsang National University, Gajwa-dong 900, Jinju, Gyeongnam 660-701, South Korea.
Mar Biotechnol (NY). 2013 Feb;15(1):56-62. doi: 10.1007/s10126-012-9456-1. Epub 2012 May 3.
To develop a multi-antigen-specific immunoglobulin new antigen receptor (IgNAR) variable (V) region phage display library, CDR3 in the V region of IgNAR from banded houndshark (Triakis scyllium) was artificially randomized, and clones specific for hen egg white lysozyme (HEL) were obtained by the biopanning method. The nucleotide sequence of CDR3 in the V region was randomly rearranged by PCR. Randomized CDR3-containing segments of the V region were ligated into T7 phage vector to construct a phage display library and resulted in a phage titer of 3.7 × 10(7) PFU/ml. Forty clones that contained randomized CDR3 inserts were sequenced and shown to have different nucleotide sequences. The HEL-specific clones were screened by biopanning using HEL-coated ELISA plates. After six rounds of screening, nine clones were identified as HEL-specific, eight of which showed a strong affinity to HEL in ELISA compared to a negative control (i.e., empty phage clone). The deduced amino acid sequences of CDR3 from the HEL-specific phage clones fell into four types (I-IV): type I contains a single cysteine residue and type II-IV contain two cysteine residues. These results indicated that the artificially randomized IgNAR library is useful for the rapid isolation of antigen-specific IgNAR V region without immunization of target antigen and showed that it is possible to isolate an antigen-specific IgNAR V region from this library.
为了开发一种多抗原特异性免疫球蛋白新抗原受体（IgNAR）可变（V）区噬菌体展示文库，我们对带纹长尾鲨（Triakis scyllium）IgNAR 的 V 区中的 CDR3 进行了人工随机化，并通过生物淘选法获得了针对鸡卵清溶菌酶（HEL）的克隆。通过 PCR 对 V 区的 CDR3 核苷酸序列进行随机重排。将包含随机化 CDR3 的 V 区片段连接到 T7 噬菌体载体上，构建噬菌体展示文库，得到噬菌体滴度为 3.7×10(7)PFU/ml。对包含随机化 CDR3 插入片段的 40 个克隆进行测序，结果显示它们具有不同的核苷酸序列。通过用 HEL 包被的 ELISA 板进行生物淘选，筛选出针对 HEL 的特异性克隆。经过六轮筛选，鉴定出 9 个为 HEL 特异性克隆，其中 8 个在 ELISA 中与阴性对照（即空噬菌体克隆）相比对 HEL 具有较强的亲和力。从 HEL 特异性噬菌体克隆中推导的 CDR3 的氨基酸序列分为四种类型（I-IV）：类型 I 含有一个半胱氨酸残基，类型 II-IV 含有两个半胱氨酸残基。这些结果表明，人工随机化的 IgNAR 文库可用于快速分离无目标抗原免疫的抗原特异性 IgNAR V 区，并且表明有可能从该文库中分离出抗原特异性 IgNAR V 区。
