肺癌特异性结合多肽的体外筛选和鉴定

Screening and identifying the peptide specifically binding to lung cancer by using phage display in vitro

目的应用噬菌体随机肽库技术筛选出与肺癌细胞特异性结合的多肽。方法以人肺癌细胞NCI-H1299为靶细胞,人胚肺细胞MRC-5为吸附细胞,对噬菌体随机12肽库进行3轮全细胞减性筛选后,随机挑取噬菌体克隆进行ELISA鉴定;对亲和力较高的阳性克隆进行DNA测序并翻译为氨基酸序列;化学合成异硫氰酸荧光素标记的多肽(FITC-ZS-5),采用细胞和组织免疫荧光法鉴定FITC-ZS-5与肺癌细胞的亲和力及特异性。结果通过3轮减性筛选后,与NCI-H1299细胞结合的噬菌体克隆得到有效的富集;ELISA结果显示5号克隆对NCI-H1299细胞亲和力最高,将其命名为Phage ZS-5;测序结果显示Phage ZS-5所表达的多肽序列在国内外均未见报道,细胞及组织免疫荧光实验结果显示FITC-ZS-5对肺癌细胞及组织具有较高的亲和力和特异性。结论应用噬菌体随机肽库技术筛选到肺癌靶向性多肽ZS-5,为肺癌的靶向治疗和诊断奠定基础。

Aim In vitro phage display was used to screen and identify a polypeptide specifically targeting lung cancer cells.Methods The lung cancer NCI-H1299 cell line and the normal lung MRC-5 cell line were used for the subtractive screening in vitro with a phage display-12 peptide library at 37℃.After three rounds of panning,a group of phage clones specifically binding to the NCI-H1299 cells were obtained,then the affinity of these clones binding to the targeted cells was studied by cell-based ELISA.The phage clones with high affinity was sequenced,thus the amino acid sequence was deduced and the peptide was synthesized and identified by immunofluorescence.Results After three rounds of panning,there was an obvious enrichment for the phage clones specifically binding to the NCI-H1299 cells.A phage clone named phage ZS-5 with high specificity and affinity to NCI-H1299 cells was identified by a cell-based ELISA.More importantly,the synthetic FITC-labeled peptide ZS-5,which corresponded to the sequence of the inserted fragment of phage ZS-5,demonstrated a high specificity to lung cancer cells and tissues,but not to normal lung cell and tissue samples,or other different cancer cells.Conclusion The peptide ZS-5 specifically binding to lung cancer has been screened from phage display peptide libraries,which can be used for targeted drug delivery in therapy or in early diagnosis of lung cancer.