Experiment Of Screening Peptides Binding Specifically To Colon Cancer Cells From Phage Random Peptide Library

Objective:To screen the peptide binding specifically to the colon cancer cells using phage display peptides library and study the affinity of these peptieds to colon cancer cells.Methods:â… :Colon cancer cells LoVo were used as the target cells and human normal colon mucous epithelial cells as the absorber cells for subtraction biopanning from a c7c phage-display peptide library.â…¡:After three rounds of subtraction biopanning in vitro,20 phage clones picked randomly were examined their affinity with colon cancer LoVo cells by enzyme-linked immunosorbent assay(ELISA) to exclude false positive clones and nonspecific phages binding to target cells and controls.â…¢:The DNA of phage positive clones specific binding to colon cancer LoVo cells were extracted and sequenced to identify the consensus sequence.â…£: According to the Cell-ELISA results,one phage positive clone with high affinity was examined by immunofluorescence detection to directly show its conjugation with colon cancer LoVo cells,and identify its targeting to colon cancer LoVo cells.Results:â… :After three rounds of substraction biopanning in vitro,phages binding to the LoVo cells were enriched from 5.6×10⁶ pfu at the first round to 2.2×10⁹ pfu at the end of the third round of substraction biopanning with an increase of the 393-fold;the Recovery namely the output/input ration was also increased from 2.8×10^-5 at the first round to 1.1×10^-2 at the end of the third round of substraction biopanning.â…¡:After substraction biopanning in vitro,20 phages clones were picked randomly on Blue-white patch experiment;then Cell-ELISA analysis suggested that 7 phages,including P1,P3,P10,P13,P14,P15 and P20,could preferably bind to colon cancer LoVo cells;5 phages,including P1,P10,P13,P14 and P20,could bind specifically to colon cancer LoVo cells,instead of human normal colon mucous epithelial cells;those results illustrate that the final 5 clones are positive and specifically targeted to colon cancer LoVo cells.â…¢:The DNA of the final 5 phage positive clones specific binding to colon cancer LoVo cells were extracted and sequenced in this study,and the sequencing information show that they are abundant in PRO with 28.6%(10/35) in proportion,and that amino acid sequences have certain homology,and that 3 phage positive clones have common sequence RPM which may have correlation to colon cancer LoVo cells surface receptor motif.â…£:Cell-ELISA results showed that the OD490nm of P14 phage binding to LoVo cells is highest,thus P14 phage was chosen for further identifications;immunofluorescence detection showed that P14 could specifically target to colon cancer LoVo cells.Conclusions:â… :5 phage positive clones(P1,P10,P13,P14,and P20) binding specifically,to colon cancer were obtained from phage display peptide library in this study and they have common sequence RPMP by amino acid sequence analysis.â…¡: Phage positive clone P14 could specifically target to colon cancer LoVo cells;it may become the targeting vector to restrain colon cancer and can be further used to the development of targeting therapeutic agents against colon cancer in the future.