Department of Clinical Laboratory Medicine, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Jiefang Da Dao 1277#, Wuhan 430022, China.
Transpl Immunol. 2013 Dec;29(1-4):39-42. doi: 10.1016/j.trim.2013.06.001. Epub 2013 Jun 25.
We select the peptide mimics of blood group A antigen by a monoclonal anti-A from a phage display 15-mer peptide library. Monoclonal anti-A was used in biopanning a phage display 15-mer peptide library. After four rounds of panning, ELISA was carried out to confirm the positive phage clones. The exogenous DNAs of the positive phages were sequenced and the corresponding amino acid sequences were deduced. Both the synthesized peptide and the phage clones were used to bind to anti-A in competitive ELISA. Erythrocyte agglutination inhibition tests were carried out to determine the mimic ability of the free synthesized peptide to the natural blood group A antigen. Computer softwares were used to simulate the interaction between the peptide and anti-A. After four rounds of biopanning, the eluted phage reached an enrichment of approximately 1600 times. Thirty-seven phage clones were chosen randomly and amplified. There were eleven clones that interacted specifically with anti-A in ELISA. DNA sequencing of the inserted oligonucleotide revealed that nine clones present a same peptide - TRWLVYFSRPYLVAT (named TRW) and each of the other two clones presented a different peptide. The synthesized free peptide TRW could inhibit the interaction of both phage displayed peptide and group A red blood cell with anti-A in competitive ELISA and hemagglutination inhibition test. Both the peptide TRW and the natural group A antigen were docked into a same cavity of anti-A in a computer simulation assay. The results indicate that peptide TRW can mimic blood group A antigen. It may be used as a proxy of natural blood group A antigen in clinical application.
我们从噬菌体展示 15 肽文库中选择了一株抗-A 单克隆抗体识别的 A 血型抗原肽模拟物。用抗-A 单克隆抗体在噬菌体展示 15 肽文库中进行生物淘选。经过四轮淘选，用 ELISA 法鉴定阳性噬菌体克隆。对阳性噬菌体的外源 DNA 进行测序，并推导出相应的氨基酸序列。用合成肽和噬菌体克隆进行竞争 ELISA 试验以检测其与抗-A 的结合能力。用红细胞凝集抑制试验测定游离合成肽模拟天然 A 血型抗原的能力。用计算机软件模拟肽与抗-A 的相互作用。经过四轮生物淘选，洗脱的噬菌体富集了约 1600 倍。随机挑选 37 个噬菌体克隆进行扩增。ELISA 试验有 11 个克隆与抗-A 特异性结合。插入寡核苷酸的 DNA 测序显示，9 个克隆呈现有相同的肽 - TRWLVYFSRPYLVAT（命名为 TRW），另外 2 个克隆各呈现不同的肽。合成的游离肽 TRW 能抑制噬菌体展示肽和 A 型红细胞与抗-A 在竞争性 ELISA 和凝集抑制试验中的相互作用。在计算机模拟试验中，将肽 TRW 和天然 A 抗原分别对接入抗-A 的同一腔中。结果表明，肽 TRW 可模拟 A 血型抗原。它可能在临床应用中作为天然 A 抗原的替代物。
