鞘氨醇-1-磷酸受体-1结合肽的筛选及功能验证

Bio-panning and functional verification of peptides binding to S1P1

目的蛋白鞘氨醇-1-磷酸受体-1(sphingosine-1-phosphate receptor-1,S1P1)在血管新生中发挥着关键性作用,并在肿瘤细胞中高表达。研究通过筛选S1P1筛选胞外区结合的功能肽段,观察S1P1的抑制对血管内皮细胞及肿瘤细胞增殖的影响。方法在大肠埃希菌BL21(DE3)中融合表达S1P1的胞外区1-46肽段,亲和纯化获得了GST-S1P11-46融合蛋白,并以此目的蛋白为靶标,采用固相包被的方法对噬菌体随机展示12肽库进行筛选,挑选有高亲和力的噬菌体,经测序得到阳性噬菌体的小肽序列;随后,将小肽偶连在匙孔蓝蛋白(keyhole limpet hemocyanin,KLH)上,并通过Western blot、细胞免疫荧光、成管实验,观察偶联肽与人脐静脉内皮细胞(human umbilical vein endothelial cell,HUVEC)表达的S1P1结合能力及对HUVEC管形成的影响,MTT检测偶联肽对肝癌细胞(human hepatocellular liver carcinoma cell line,HepG2)增殖的影响。结果获得了与S1P1有较高亲和力的2种12肽。细胞实验证实偶联肽能结合细胞表面的S1P1受体,而且2种偶联肽均能抑制HepG2的增殖,并抑制HUVEC细胞的成管行为。结论利用噬菌体肽库筛选技术,成功筛选出了具有抑制作用的S1P1结合肽。

Objective Sphingosine-l-phosphate receptor-1 plays an important role in angiogenesis with high expression in tumor cells. The aim of this study is to screen the function peptides that can bind to extracellular region of S1P1 （ sphingosine-l-phos- phate receptor. Methods The fusion protein GST-S1P1146 were induced to express in E. coli BL21 （DE3）, then isolated and puri- fied by GST exchange resin. The purified proteins coated on polystyrene plate were used as targets to carry out six rounds of bio-pan- ning. The affinity of selected recombinant phage clones was tested by ELISA and the finally amino acid sequences of 12-peptides were detected. Three 12-peptides were synthesized and then coupled to KLH. We certified the binding capacity between S1P1 and the cou- pled peptides and the influence of HUVEC tube formation by Western blot and immunofluoresence tube formation experiments, MTT was employed to examine the influence of coupled peptides on HepG2 cell proliferation. Results The obtained two kinds of 12-pep- tide can specifically bind to S1P1 ; cytological experiments confirmed that both of them could inhibit HepG2 proliferation and prevent HUVEC cells gathering into tubes. Conclusion By using the triage techniques of phage peptide library, we successfully screened the S1P1 binding peptides with the capacity of inhibition.