SGC7901-VCR细胞特异性内化噬菌体多肽的筛选和初步鉴定

Screening and identification of phage polypeptides internalized into SGC7901-VCR CELL

目的筛选能够特异性内化入SGC7901-VCR耐药细胞的噬菌多肽,为肿瘤的靶向性药物治疗提供实验基础。方法以SGC7901-VCR耐药细胞为靶分子对噬菌体12肽库进行全细胞筛选,将第三轮内化入细胞内的噬菌体扩增后分别测定其在SGC790I-VCR细胞中不同时段的生存率,挑选能够抗细胞内蛋白酶降解的噬菌体单克隆,用细胞化学的方法对其内化结果进行鉴定,并通过竞争性内化实验进一步挑选能够特异性内化入SGC7901-VCR细胞的噬菌体多肽。结果通过三轮淘洗,内化噬菌体的回收率从2.9×10-5%增加到9.3×10-2%,阳性克隆得到富集;随机挑取4个内化入细胞24小时的噬菌体单克隆,细胞化学证实其中有两个单克隆可内化入细胞,竞争性内化实验表明这两个单克隆内化效率均较高。结论这两个多肽可能会作为载体将化疗药物及其它小分子转运入SGC7901-VCR细胞内。

Objective To screen the phage polypeptides specifically internalized into SGC7901 - VCR and provide basis for targeted drug delivery in amacr cells. Methods Biopanning the Ph. D. 12 library with whole SGC7901 - VCR cell. After deactivation of cell surface bound phages , internalized phages were titered. The surviving phages were recovered at the indicated time points by cell lysis, and the survival rates of the phages were calculated. Internalized phages resisting endosomal degradation were selected randomly and internalized result were identified by cell chemistry. Competivtive internalized efficiency of them were tested. Results After 3 rounds, the recovery rate of internalized phages were from 2.9×10^- 5 to 9.3 ×10^-2, which means that specific enrichnent had been achieved. Four phage clones which had been internalized into SGC7901- VCR for 24 hours were selected randomly and cell chemistry showed the two of them can internalized into SGC7901- VCR. The two phage clones had higher internalized efficiency. Conclusion The two highly specific polypeptides may be vectors to delivery drugs to the SGC7901- VCR cell.