噬菌体展示技术筛选胃癌高表达CD44分子特异性多肽序列的可行性分析

Feasibility of phage-display technology in screening specific polypeptide sequences in gastric cancer cells with high expression of CD44

目的探讨噬菌体展示技术筛选胃癌高表达CD44分子特异性多肽序列的可行性。方法使用噬菌体展示随机十二肽文库,以CD44纯化蛋白为筛选靶点,进行4轮亲和力筛选。从第四轮的洗脱物中随机挑选30个噬菌体克隆,对这些克隆进行测序并对序列进行生物信息学分析。通过ELISA法检测噬菌体克隆对CD44分子的亲和力,挑选阳性克隆,再通过细胞免疫荧光法进一步鉴定阳性克隆。结果从第4轮的洗脱物中随机挑选的30个噬菌体克隆中共有7种多肽序列,重复20次六肽基序WHXXXX。该基序与CD44透明质酸结合域能很好地结合,ELSIA法挑选出S1、S2、S5、S7四个阳性克隆。细胞免疫荧光法鉴定出对CD44亲和力最好的克隆为S1-WHXXXXXXQQA。结论通过噬菌体展示技术筛选S1-WHXXXXXXQQA多肽序列对CD44的亲和力和特异性最好;该序列有望成为特异性探针用于胃癌的分子影像学诊断。

Objective To investigate the feasibility of phage-display technology in screening the specific polypeptide sequences in the gastric cancer cells with high expression of CD44.Methods A phage-displayed random twelve-peptide library was used to screen CD44 purified protein for four rounds of affinity screening.Thirty clones were selected and were sequenced based on the single-strained DNA.ELISA was used to test the binding affinity of the selected phages to CD44.The affinity of positive clones to CD44 integrated to the cell membrane was detected by immuno uorescence assay.Results During the four rounds,30 clones were selected from the eluate of the 4th round of sequencing,and 7 sequences were determined.A six peptide motif which repeated 20 times was found in following analysis.Among the 7 sequences,4 positive clones(S1,S2,S5 and S7)were identified by ELISA.The phage inserted sequence S1-WHXXXXXXQQA was identified,which had the highest affinity and specificity to CD44.Conclusion Through phage-display technology,S1-WHXXXXXXQQA peptide sequence has the best affinity and specificity to CD44,which may be a novel molecular probe for the molecular imaging diagnosis of gastric cancer.