论文部分内容阅读
目的筛选人源肝癌组织特异性结合短肽。方法体外培养人源肝癌细胞株BEL-7402,建立荷瘤裸鼠模型。尾静脉注射噬菌体12肽文库至荷瘤裸鼠体内,循环20 m in后回收肿瘤组织中噬菌体,同时取正常对照组织进行噬菌体效价测定和免疫组化观察。将回收的噬菌体扩增、纯化,并以此作为起始物进行下一轮筛选。经过3轮体内筛选,得到与肝癌组织或细胞特异结合的肽段。随机挑选噬菌体单克隆进行测序,分析序列同源性后进行体外细胞酶联免疫吸附试验(ELISA)和体内回输实验,验证噬菌体克隆的导向性。结果经过3轮体内筛选,瘤组织中噬菌体的回收率逐步提高,回收量随着输入量的增加迅速增加,而每轮筛选肝组织中的噬菌体回收量始终保持在一个恒定范围,并不随输入量的增加而增加。免疫组化结果显示,第3轮筛选后,瘤组织中的噬菌体得到高水平富集,同时其他组织的非特异性结合降至最低。肝癌细胞特异性结合最强的是A54号单克隆,A67、B2号单克隆次之。B2的导向效果最好,A54次之。通过对噬菌体单克隆的序列分析,初步确定了PSS/PTT基序。结论利用体内噬菌体展示技术,可以成功筛选到与肝癌细胞或组织特异结合的噬菌体肽。
Objective To screen human liver cancer tissue - specific binding peptides. Methods Human hepatoma cell line BEL-7402 was cultured in vitro to establish a tumor-bearing nude mouse model. The phage 12 peptide library was injected into the tail vein of tumor-bearing nude mice, and the phage were recovered from the tumor tissue after circulating for 20 min, and the phage titer and immunohistochemistry were also observed in normal control tissues. The recovered phage was amplified, purified and used as a starting material for the next round of screening. After three rounds of in vivo screening, peptides that specifically bind to liver cancer tissues or cells are obtained. Randomly selected phage clones were sequenced. Sequence homology was analyzed by in vitro cell enzyme-linked immunosorbent assay (ELISA) and in vivo reinfusion experiments to verify the targeting of phage clones. Results After three rounds of in vivo screening, phage recovery gradually increased in the tumor tissue, and the recovery rapidly increased with the increase of the input amount. The recovery of phage in each round of the screening of liver tissue remained constant within a constant range, Increase and increase. Immunohistochemistry showed that after the third round of screening, the phage in the tumor tissue was highly enriched, while the nonspecific binding of other tissues was minimized. The most specific binding of hepatoma cells was monoclonal A54, followed by monoclonal A67 and B2. B2 the best guidance, A54 second. Through the sequence analysis of the phage monoclonal, the PSS / PTT motif was preliminarily determined. Conclusion The phage display peptide can be successfully screened by using phage display technology in vivo.