从噬菌体展示十二肽库中筛选钠泵α_1亚基M1-M2膜外区特异性结合肽

Screening of Peptides That Specifically Bind to Sodium Pump α_1 Subunit M1-M2 Extramembrane Domain from Phage Display 12 Peptide Library

目的获得人钠泵α1亚基M1-M2膜外区的特异性结合肽,以阻断或拮抗哇巴因对钠泵的抑制作用,为高血压的治疗提供理论和实验依据。方法以人工合成的钠泵α1亚基M1-M2膜外区多肽(ATEEEPQNDN-LGGGS)为靶分子,从噬菌体随机12肽库淘选能与之特异性结合的多肽,通过双夹心酶联免疫吸附试验(ELISA)和浓度依赖性实验鉴定噬菌体阳性克隆,提取DNA测序,推导其氨基酸序列,并检测其对哇巴因的竞争性拮抗作用。结果从噬菌体12肽库中筛选出4个阳性噬菌体克隆,测序后其氨基酸序列完全一致,均为:WHWRNPDF-WYLK。哇巴因竞争性抑制试验显示,获得的噬菌体展示肽可以与哇巴因竞争钠泵α1亚基M1-M2膜外区结合表位,阻止哇巴因与钠泵结合。结论获得了人钠泵α1亚基M1-M2膜外区的特异性结合肽,它能竞争性抑制哇巴因与钠泵的结合,为进一步探讨钠泵的作用机制及高血压的治疗奠定了基础。

Objective To obtain specific binding peptides to human M1-M2 extramembrane domain of sodium pump α1 subunit for antagonization or blockage action of ouabain on sodium pump;to provide theoretical and experimental bases for hypertension therapy.Methods Screen the specific binding peptides from phages that display random peptide libraries using synthetic M1-M2 subunit extramembrane domain peptide （ATEEEPQNDNLGGGS） of sodium pump α1 as the target molecules.Monoclonal phages were picked up randomly and identified by double-sandwich ELISA and concentration-dependent experiments.The phage ssDNA of positive clones was extracted,sequenced,and the corresponding amino acid sequences were deduced.Ouabain competitive inhibition test was performed to detect the competitive inhibition effect of phage-displayed peptide on ouabain.Results Four positive phage clones were selected from phages display random peptide libraries.Sequencing analysis showed that the amino acid sequences of the four phage clones are identical,i.e.WHWRNPDFWYLK.Ouabain competitive inhibition test showed that the displayed peptides can bind specifically to M1-M2 extramembrane domain,can block or antagonize the binding of ouabain to sodium pump.Conclusion A specific binding peptide of human sodium pump α1 subunit M1-M2 extramembrane domain was obtained from phage-displayed random peptide library.The peptide can competitively inhibit the binding of ouabain to sodium pump,which laid a foundation for further exploring the functions of sodium pump and for better treatments of hypertension.