肝癌细胞特异性内在化肽的筛选和初步鉴定

Selection and preliminary identification of hepatocarcinoma-binding and internalizing peptide

应用噬菌体肽库技术筛选与肝癌细胞特异性结合并可以内在化的短肽,为肝癌的导向治疗奠定基础。以肝癌细胞株BEL-7404作为筛选靶细胞,对噬菌体展示随机12-肽库进行亲和淘选。经过3轮淘选后,共随机挑选出20个噬菌斑进行扩增和测序,同时用细胞ELISA检测其与肝癌细胞的结合情况,并通过免疫荧光术、流式细胞术等方法进一步鉴定噬菌体克隆对肝癌细胞的导向性及内在化的效果。结果表明3轮淘选所得的噬菌体回收率逐步提高,显示淘选过程对随机肽库有一定的富集效果。从扩增的噬菌体克隆中选择结合力最强的LJ08通过免疫荧光术、流式细胞术鉴定,结果显示LJ08与肝癌细胞呈特异性结合并内在化进入肝癌细胞。结论认为,通过对噬菌体随机肽库的筛选,得到可以与肝癌细胞BEL-7404结合并可以内在化的噬菌体肽,为该肽作为肝癌导向治疗的药物载体奠定基础。

Phage display peptide libraries had been successfully used to screen hepatocarcinoma-binding and internalizing peptides, With great potential to be used in targeted drug therapy for liver cancer. In the present study the phage display 12-mer peptide library was screened with liver cancer cell line BEL-7404, and after three rounds of biopanning, 20 positive clones were picked up and sequenced. Their affinity to BEL-7404 was identified by cell-based ELISA. Then, the clone LJ08 were chosen for further identification by immunofluorescence and FACS. It was demonstrated that the phage titers were significantly improved after each round of biopanning. The results of cell-based ELISA indicated that LJ08 had the highest affinity to BEL- T404. Besides, LJ08 were identified efficiently endocytosed into BEL-7404 cells, but not the normal cell line as demonstrated by immunofluorescence and FACS. It is feasible to screen and isolate targeted peptides that bind specifically to and internalizing into BEL-7404 cells using phage display peptide libraries. These results may lay a foundation for LJ08 to become a drug carrier used for the targeted therapy of hepatocellular carcinoma.