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目的:体外差减筛选噬菌体展示环七肽库,获得与中国人膀胱癌BIU-87细胞系高度结合的小分子多肽并鉴定其结合特异性。方法:以中国人膀胱癌BIU-87细胞作为靶细胞,正常人膀胱上皮细胞为吸附细胞,对噬菌体展示环七肽库进行3轮体外差减筛选。ELISA法鉴定与BIU-87细胞呈强阳性结合的噬菌体克隆,对其编码的DNA进行序列测定和同源性分析。化学合成与BIU-87细胞强阳性结合的肽段并制备成FITC标记的荧光探针,流式细胞术、荧光显微镜下鉴定其与BIU-87细胞、正常人膀胱上皮细胞、人前列腺癌PC3M细胞、人肝癌SMMC-7721细胞以及人结肠癌HCT116细胞的特异结合能力。结果:经3轮差减筛选将噬菌体展示环七肽库富集了25倍,阳性率达76%。共获得10个强阳性克隆,DNA共有序列为SISSLTH、MARYMSA、TVRTSAD。BIU-87细胞与小分子荧光探针FITC-SISSLTH的结合率为(80.06±8.78)%,显著高于FITC-MARYMSA的(52.93±7.28)%、FITC-TVRTSAD的(38.04±7.47%)、FITC-EDRKETA的(1.91±1.37)%和FITC的(9.85±2.9)%(均P<0.01)。FITC-SISSLTH与BIU-87细胞的结合率显著高于与正常人膀胱上皮细胞、人前列腺癌PC3M细胞、人肝癌SMMC-7721细胞和结肠癌HCT116细胞的结合率[(80.06±8.78)%vs(13.89±1.97)%,(8.13±2.85)%,(27.00±2.87)%,(2.33±1.75)%;均P<0.01]。结论:噬菌体展示环七肽库经3轮体外差减筛选获得高效结合BIU-87细胞的导向肽SISSLTH,具有良好的结合特异性。
OBJECTIVE: To screen the phage display library of cycloheptenin by differential subtraction in vitro and to obtain small molecule peptides highly binding to Chinese bladder cancer cell line BIU-87 and to identify its binding specificity. Methods: Human bladder cancer BIU-87 cells were used as target cells, normal human bladder epithelial cells were adsorbed cells, and the phage displayed cyclic heptapeptide library was subjected to three rounds of in vitro differential screening. The phage clones strongly binding to BIU-87 cells were identified by ELISA, and the DNAs were sequenced and analyzed for homology. Peptide with strong positive binding to BIU-87 cells was chemically synthesized and labeled with FITC-labeled fluorescent probe. Flow cytometry and fluorescent microscopy were used to identify the expression of BIU-87 cells, normal human bladder epithelial cells, human prostate cancer PC3M cells , Human hepatoma SMMC-7721 cells and human colon cancer HCT116 cells. Results: After three rounds of subtractive screening, the phage display peptide library enriched 25 times, the positive rate of 76%. A total of 10 strong positive clones were obtained. DNA consensus sequences were SISSLTH, MARYMSA and TVRTSAD. The binding rate of BIU-87 cells to FITC-SISSLTH was (80.06 ± 8.78)%, which was significantly higher than that of FITC-MARYMSA (52.93 ± 7.28)% and FITC-TVRTSAD (1.91 ± 1.37)% of EDRKETA and (9.85 ± 2.9)% of FITC (all P <0.01). The binding rate of FITC-SISSLTH to BIU-87 cells was significantly higher than that of normal human bladder epithelial cells, human prostate cancer PC3M cells, human hepatoma SMMC-7721 cells and colon cancer HCT116 cells [(80.06 ± 8.78)% vs 13.89 ± 1.97%, 8.13 ± 2.85%, 27.00 ± 2.87%, 2.33 ± 1.75% respectively; all P <0.01]. CONCLUSION: The phage displayed loop heptapeptide library has been screened by three rounds of in vitro differential in vitro to obtain the targeting peptide SISSLTH which binds to BIU-87 cells with good binding specificity.