噬菌体肽库在筛选及鉴定高转移性肝癌特异性结合肽中的应用及其意义

Application of phage-display peptide library in screening short peptides binding specifically to high metastatic potential hepatocellular carcinoma cells and its significance

目的:探讨噬菌体环七肽库在筛选与肝癌高转移细胞株HCCLM3特异高效结合的短肽中的应用,为进一步探索肝癌转移的分子机制和寻找肝癌转移标志物奠定基础。方法:以人肝癌高转移细胞株HCCLM3为靶细胞、人肝癌低转移细胞株SMMC7721为吸附细胞对噬菌体环七肽库进行4轮差减筛选,采用ELISA方法进一步筛选出与HCCLM3细胞高度亲和的阳性克隆(即实验组A450nm值高于对照组3倍以上),并利用免疫细胞化学方法鉴定噬菌体阳性克隆的特异性并测序。结果:经过4轮陶筛后,噬菌体在筛选靶细胞上出现明显富集,且逐轮提高(P<0.05);利用ELISA法对筛选后随机挑取的20个噬菌体克隆进行初步鉴定,得到6个能与肝癌细胞HCCLM3亲和度较高的阳性克隆(C4、C6、C7、C10、C11和C17);通过免疫细胞化学染色鉴定阳性克隆靶向肝癌细胞HCCLM3的特异性,与对照组比较,C7噬菌体对高转移潜能肿瘤细胞具有较高特异性(P<0.05);测序显示6个阳性克隆氨基酸序列无同源性。结论:利用噬菌体环七肽库筛选得到与人肝癌高转移细胞株HCCLM3具有较高亲和力的多肽。

Objective To explore the application of c7c phage-display peptide library in screening the short peptides which bind specifically to high metastatic potential hepatocellular carcinoma cells HCCLM3, and to lay foundation for research on the molecular mechanism of hepatoma metastasis and detection of the markers of hepatoma metastasis. Methods The human high metastatic potential hepatocellular carcinoma ceils HCCLM3 were used as the target cells and the human low metastatic potential hepatocellular carcinoma cells SMMC7721 as the absorber cells for subtraction biopanning from c7c phage-display peptide library. The affinity and specificity of the positive phage clones were identified by ELISA and immunocytochemistry, and the amino acid sequences were deduced by DNA sequencing. Results After 4 rounds of screening, the phages which can bind tO the HCCLM3 cells were enriched obviously （P〈0.05）. 6 positive phage clones （C4, C6, C7, C10, Cll and C17） with high affinity were identified by ELISA. After cell immunocytochemistry, 1 peptide namely C7 with higher specificity to HCCLM3 cells compared with negative control was found （P〈0. 05） and no conserved motif was found in these peptides. Conclusion The peptides specifically binding to HCCLM3 cells are screened and identified from c7c phage-display peptide library.