利用体内噬菌体展示技术筛选肝癌组织特异性结合肽

Selection of the peptides specifically binding to hepatoma by using phage display in vivo

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 the peptides binding to hepatoma specifically. Methods Nude mice were inoculated with human tumor cells BEL-7402, then the Ph. D. -12 Phage Displa〉 Peptide Library was injected intravenously （tail vein） into mice, After 20min the mice were sacrificed and the phage rescued from tumor tissues. All the tissues shouht be made an appraisal using immunohistochemistry and titefing, The phage recovered from the tissues were amplified and purified then re-injected for next round screening. After screening for 3 rounds in vivo, the phage-peptides that homed to the tumor tissues or cells were obtained. Then these phage clones were sequenced to analyze the motif. All the sequenced clones were appraised by using cell ELISA and titering the distribution in rico. Results Through the appraisal from vivo and vitro and peptides＇ sequences, several target motifs were preliminarily determined. Conclusions Some phage-peptides which could specifically bind tumor cells or tissues can be selected successfully from the random twelve-peptide library by means of phage display in vivo.