摘要
目的本研究对噬菌体展示进行改良,拟获取对Nanodiscs组装即体外膜蛋白模拟后的CD44v6具有结合力及特异性的多肽配体序列。方法对真核来源CD44v3-v10蛋白进行Nanodiscs包被,采取SDS-PAGE、分子排阻色谱验证。以Nanodiscs组装后的CD44v3-v10蛋白为靶蛋白,采用液相筛选磁珠吸附及抗体特异性竞争洗脱的方法获得针对于CD44v6的候选噬菌体,测序获得多肽配体序列。用ELISA法以及噬菌体洗脱试验验证并选择最佳序列。结果SDS-PAGE显示Nanodiscs包被后出现新增条带,分子排阻色谱提示产物峰形成。噬菌体筛选过程中出现明确的富集效应,测序后发现30个克隆包含6条序列。噬菌体ELISA结果显示待测克隆和靶蛋白结合的OD值,与待测克隆和对照蛋白结合、对照克隆和靶蛋白结合的OD值的差异存在统计学意义(P<0.05)。选取OD值最高的3个克隆进行噬菌体洗脱试验。待测克隆与CD44v6阳性的SGC-79细胞结合数量,与待测克隆与CD44v6阴性的HEK-293细胞结合,对照克隆与SGC-7901细胞结合数量间差异存在统计学意义(P<0.05)。选取与阳性细胞结合数量最多、阴性细胞结合数量少的噬菌体克隆(携带多肽序列HNTYVTSFHRNY)为最佳克隆。结论本研究成功对CD44进行Nanodiscs包装,并利用其筛选出CD44v6特异性多肽序列。
Objective To screen the peptide ligand binding and specific to CD44v6 using modified phage display based on assembled nanodiscs that can help imitate the active form on cell membrane of target protein.Methods CD44v3-v10 eukaryotic recombinant protein was assembled in nanodiscs.The protein-nanodiscs were convinced with SDS-PAGE and size-exclusion chromatogrphy.Routine phage display strategy was modified based on nanodisc assembled CD44v3-v10 in solution with magnetic beeds and antibody competitive elution.After sequenced,the candidate phages were selected and defined with ELISA and phage elution examination.Results New band and product peak occurred in SDS-PAGE and size-exclusion chromatogrphy examination separately after nanodiscs assembly.Phages enriched during the panning process.Six individual sequences existed in 30 selected clones.Results of phage ELISA exhibited that OD values of candidate phage binding to target protein was significantly different from that of control phage binding to target protein and that of candidate phage binding to control protein(P<0.05).Phage elution examination also showed that the binding ability of candidate phages on CD44v6 positive SGC-7901 was more than that of control phages on SGC-7901 cell and that of candidate phages on CD44v6 negative cells(P<0.05).The phage clone,with displayed peptide HNTYVTSFHRNY,was chosen as the best due to its binding activity and selectivity to target protein.Conclusion CD44 protein is assembled in nanodiscs successfully,which can be used to screen and define the CD44v6 specific peptide.
作者
张丹
卢桂芳
冯云
刘亚萍
赵倩
任牡丹
卢新兰
和水祥
ZHANG Dan;LU Guifang;FENG Yun;LIU Yaping;ZHAO Qian;REN Mudan;LU Xinlan;HE Shuixiang(Department of Gastroenterology,First Affiliated Hospital of Xi’an Jiaotong University,Xi’an 710061,China)
出处
《山西医科大学学报》
CAS
2020年第7期647-653,共7页
Journal of Shanxi Medical University
基金
国家自然科学基金青年基金资助项目(81602611)
陕西省重点研发计划项目(2017-SF225,2017-SF255)。
