噬菌体随机肽库对肝癌细胞特异性结合短肽的筛选及鉴定

Screening and identification of hepatocellular carcinoma cell specific binding peptides using phage display peptide library

目的:通过噬菌体随机肽库,筛选人肝癌细胞结合短肽,并对其与肝癌细胞的特异结合能力进行生物学鉴定.方法:以L-02为阴性消减细胞,HepG2为靶细胞,对噬菌体随机七肽库进行4轮消减筛选.并随机挑取26个阳性噬菌体克隆进行序列测定及同源性分析.以ELISA法鉴定噬菌体与HepG2细胞的结合活性.通过免疫细胞化学染色、免疫组织化学染色鉴定H3噬菌体克隆与肝癌细胞结合的特异性.人工合成短肽HBP3,利用免疫荧光技术观察其与肝癌细胞的结合特异性.结果:4轮筛选使噬菌体在靶细胞HepG2上出现明显富集.所挑取的26个阳性噬菌斑,经测序得到各噬菌体单克隆的多肽插入序列,最终产生5条氨基酸序列.经BLAST分析发现,在蛋白质数据库中未发现与这些短肽序列具有较好同源性的蛋白质分子.ELISA分析鉴定显示,5个阳性克隆在HepG2和L-02细胞上有差异性结合,其中H3噬菌体克隆对两种细胞的结合差异性最大.免疫细胞化学染色及免疫组织化学染色均显示,H3噬菌体克隆对肝癌细胞的结合靶向性明显高于对照细胞.免疫荧光显色证实,FITC-HBP3能特异性地结合于肝癌细胞的胞膜及核周胞质.结论:H3噬菌体克隆所呈现的七肽HBP3能与靶细胞高亲合性结合,为进一步研制用于治疗肝癌的高靶向性药物奠定实验基础.

AIM： To screen the peptides binding specifically to the hepatoma cells using phage display peptides library and identify their affinity. METHODS： Whole-cell subtractive screening with phage display 7^TM peptide library was performed on HepG2 cells and normal L-02 cells. After 4 rounds of panning in vitro, 26 phage clones picked randomly were sequenced to identify the consensus sequence. The affinity of 5 phages with hepatoma cells was examined by ELISA assay. The immunocytochemical and immunohistochemical stainings were performed to determine the specificity of the phages to hepatoma cells. Peptides displayed on the phages were synthesized and the immunofluorescence microscopy was used to study the binding of them to hepatoma cells. RESULTS： After panning, phages binding to the HepG2 cells were enriched. ELISA results suggested that 5 phages binded differentially to hepatoma cells and control cells. The immunocytochemical and immunohistochemical stainings showed that H3 phage preferably binded to bepatoma cells rather than controls, and phage was also found to be able to bind to hepatoma tissue sections. Using immunofluorescence microscopy, fluorescence labeled HBP3 peptide was seen on the membrane and in the perinuclear cytoplasm of hepatoma cells. CONCLUSION： HBP3 has higher affinity with its target cells and may become the target molecular to hepatoma therapy in the future.