噬菌体展示技术筛选人乳腺髓样癌细胞特异性结合肽

Selection of peptide specifically binding to human mammary medullary carcinoma cells by phage display

目的利用噬菌体随机七肽库体外筛选与人乳腺髓样癌Bcap-37细胞特异性结合的小分子多肽。方法培养人乳腺髓样癌Bcap-37细胞作为靶细胞,人正常乳腺上皮细胞MCF-10A为吸附细胞,将噬菌体展示随机七肽库与人乳腺髓样癌Bcap-37细胞进行减性体外筛选,用ELISA法鉴定噬菌体对人乳腺癌Bcap-37细胞的亲和力。提取亲和力较高的单克隆噬菌体DNA,测定DNA序列并翻译出相应的氨基酸序列,进行同源性分析。结果利用噬菌体随机七肽库通过4轮减性筛选获得了与人乳腺髓样癌Bcap-37细胞特异结合的小分子多肽。ELISA法表明,第1,3,5,6,8,12,14,16,17,18和20号单克隆噬菌体与人乳腺髓样癌Bcap-37细胞有很强的亲和力和特异性。测序结果显示,重复性最高的序列为LXRNTNX。结论利用噬菌体展示技术体外筛选获得了能与人乳腺癌Bcap-37细胞特异性结合的小分子多肽LXRNTNX,经检索该肽段与已知蛋白尚无同源性,因此它很有可能成为乳腺癌的早期诊断和靶向治疗新的靶点。

Objective To screen small molecular peptides in vitro that can specifically bind to human mammary medullary carcinoma Bcap-37 cells using Phage Display Random Heptapeptides Library.Methods Human mammary medullary carcinoma Bcap-37 cells were cultured as target cells,and human mammary epithelial cells MCF-10A were chosen as absorption cells.The Phage Display Random Heptapeptide Library was used for four rounds subtraction biopanning in vitro with human mammary medullary carcinoma Bcap-37 cells.The affinity of phage to human breast cancer Bcap-37 cells was determined by ELISA.The monoclonal phage DNA with high affinity was extracted and sequenced to identify the gene sequence and the corresponding amino acid sequences,and analyze the homology of amino acid sequence.Results After four rounds subtraction biopanning,the phages specifically binding to Bcap-37 cells were highly enriched.The monoclonal phage1,3,5,6,8,12,14,16,17,18 and 20 had strong affinity and specificity with human mammary medullary carcinoma Bcap-37 cells.Sequencing results showed the highest repeat sequences was LXRNTNX.Conclusion The small molecular polypeptide LXRNTNX that can specifically bind to human breast cancer Bcap-37 cells is successfully screened in vitro using phage-display technology.After retrieval,the peptide segment is not homologous with known proteins,so it should be likely to become a new target for early diagnosis and targeted treatment of breast cancer.