中国人膀胱癌BIU-87细胞导向肽的体外筛选与特异性鉴定

Screening and identifying of homing peptides to bladder cancer BIU-87 cells in Chinese

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 a peptide strongly binding to the bladder cancer BIU-87 cells from phage 7-mer cyclic peptide library in vitro in Chinese, and identify its specificity. Methods： 3 rounds subtraction screening was performed on a phage 7-mer cyclic peptide library, with the BIU-87 cells as the target cells and normal human bladder epithelial cells as the absorber cells. Phage clones which can positively bind to BIU-87 cells were identified by ELISA, and those coding DNA were sequenced to analyze the homology. Strongly positive peptide was chemically synthetized to prepare fluorescent probe by taging fluorescein isothiocyanate （FITC）. The affinity between the fluorescent probes and BIU- 87 bladder cancer cells, human normal bladder epithelial cells, human prostate carcinoma PC3M cells, human hepatocellular carcinoma SMMC-7721 cells and human colon cancer HCT116 cells were identified by flow cytometry and fluorescence microscopy.Results：The c7c phage-display peptides library was enriched for 25 times through 3 rounds of subtraction screening and the positive rate was 76%. 10 strongly positive clones were obtained. For the 10 strongly positive clones, the amino acid sequences of SISSLTH, MARYMSA and TVRTSAD were consensus sequence. The binding rate of small molecular fluorescent probe FITC-SISSLATH binding to the BIU- 87 bladder cancer cells （80.06±8.78）% was obviouly higher than of FITC-MARYMSA（52.93±7.28）%, FITC-TVRTSAD（38.04±7.47）%, FITC -EDRKETA（1.91±1.37）% and FITC（9.85±2.9）% （all P〈0.01）. The binding rate of the BIU- 87 bladder cancer cells binding to FITC-SISSLATH（80.06±8.78）% was higher than that of the normal human bladder epithelial cells （13.89±197）%, human prostate carcinoma PC3M cells（8.13±2.85）%, human hepatocellular carcinoma SMMC-7721 cells （27±2.87）% and human colon cancer HCT116 cells（2.33±1.75）%（all P〈0.01）. Conclusion： A targeting peptide SISSLTH binding to BIU-87 cells with high specificity and efficiency was obtained from the phage 7-mer cyclic peptide library through 3 rounds of subtraction screening in vitro.