Yau Kerrm Y F, Groves Maria A T, Li Shenghua, Sheedy Claudia, Lee Hung, Tanha Jamshid, MacKenzie C Roger, Jermutus Lutz, Hall J Christopher
Department of Environmental Biology, University of Guelph, Guelph, ON N1G 2W1, Canada.
J Immunol Methods. 2003 Oct 1;281(1-2):161-75. doi: 10.1016/j.jim.2003.07.011.
Picloram-specific variable fragments (V(HH)s) of heavy chain antibodies (HCAbs) were selected from a nai;ve-llama library using ribosome display technology. A cDNA library of V(HH)s was constructed from lymphocytes of a non-immunized llama and engineered to allow in vitro transcription and translation. With no stop codons present on the transcripts, trimeric complexes of ribosomes, mRNAs and nascent peptides were produced for affinity selection, i.e. panning. After three cycles of panning, seven different V(HH)s all belonging to the V(HH) subfamily 1 were isolated. Following another three cycles of selection, only two of the seven V(HH)s persisted. A comparison of these two sequences with known sequences in the literature suggests that point mutations may have been introduced into the DNA pool during PCR amplification steps of library construction, panning and/or cloning. Three separate point mutations causing three independent amino acid changes (nonsynonomous mutations) accumulated in the same sequence and enriched throughout the selection protocol, suggesting that these changes confer binding advantages. Surface plasmon resonance (SPR) analysis was used to determine binding kinetics of the two clones (3-1D2 and 3-1F6) representing the two different sets of isolated complementarity determining region (CDR)3s. Measured K(D)s were 3 and 254 muM, respectively. The results indicate that ribosome display technology can be used to efficiently isolate hapten-specific antibody (Ab) fragments from a nai;ve library and concurrently introduce diversity to the selected pool thereby facilitating molecular evolution. Ribosome display technology can compensate for the limited diversity of a V(HH) nai;ve library and provide an unlimited source of affinity-matured immunoactive reagents in vitro.
使用核糖体展示技术从一只未经免疫的美洲驼的天然文库中筛选出了针对毒莠定的重链抗体（HCAbs）的特异性可变片段（V(HH)s）。从一只未免疫的美洲驼的淋巴细胞构建了V(HH)s的cDNA文库，并进行工程改造以实现体外转录和翻译。由于转录本上不存在终止密码子，因此产生了核糖体、mRNA和新生肽的三聚体复合物用于亲和选择，即淘选。经过三轮淘选，分离出了七个不同的V(HH)s，它们都属于V(HH)亚家族1。在另外三轮选择之后，七个V(HH)s中只有两个留存下来。将这两个序列与文献中的已知序列进行比较表明，在文库构建、淘选和/或克隆的PCR扩增步骤中，可能已将点突变引入DNA文库中。三个单独的点突变导致了三个独立的氨基酸变化（非同义突变），这些突变在同一序列中积累并在整个选择过程中富集，表明这些变化赋予了结合优势。使用表面等离子体共振（SPR）分析来确定代表两组不同的分离互补决定区（CDR）3的两个克隆（3-1D2和3-1F6）的结合动力学。测得的解离常数（K(D)s）分别为3和254 μM。结果表明，核糖体展示技术可用于从天然文库中高效分离半抗原特异性抗体（Ab）片段，并同时为所选文库引入多样性，从而促进分子进化。核糖体展示技术可以弥补V(HH)天然文库多样性的不足，并在体外提供无限的亲和力成熟的免疫活性试剂来源。
