Phage Display Peptide Library For Screening The Peptides That Specifically Bind To Human Osteoblasts Cells

To obtain short peptide which bind specifically to human osteoblasts by meansof screening from12-mer peptide phage-display library through phage displaytechnology, which will provide the basis of the experiment of the Ti surfacebiolization modification.The whole study consists of two parts as follows:PartⅠ Phage display peptide library for screening the peptides that specificallybind to osteoblasts cellsHuman Calvarial osteoblasts were used as the target cells for whoie-cellbiopanning from a12-mer peptide phage-display library. Cell eluent and cell lysisbuffer were cultivate and count respectively after washing. Forty six phagemonoclonals (consist of23samples of cell eluent and23samples of cell lysis)wererandomly selected from counting tablet, which were analysised by enzyme-linkedimmunosorbent assay (ELISA) and immunofluorescence detection to authenticatethe positive phage clones by human gingival fibroblast as the absorber cells. Thepositive phage clones were deduced by DNA sequencing.Twenty two positive phage clones were found out, whose specific binding forceagainst human osteoblasts were more than2times that of human gingival fibroblastscells. The same specific peptides were found out from cell eluent and cell lysis,whose ratio were about the same. This study indicated that the specific phage againsthuman osteoblasts were bind to cells by the peptides which displayed on the surfaceof themself. The phage particles can internalize the cells by endocytosis which weremediated vie this receptor. Amino acid sequence of the highest frequency peptidewas MGWSW WPETWPM, whose amino acid composition and their basic biochemical properties of short peptide were analyzed.PartⅡ Phage display peptide library for subtractive screening the peptides thatspecifically bind to osteoblasts cellsHuman Calvarial osteoblasts were used as the target cells and human gingivalfibroblasts as the absorber cells for subtraction biopanning from a12-mer peptidephage-display library through phage display technology. Cell eluent were cultivateand count after washing. Thirty phage monoclonals were randomly selected fromcounting tablet, which were analysised by enzyme-linked immunosorbent assay(ELISA) and immunofluorescence detection to authenticate the positive phage clonesby human osteoblasts and human gingival fibroblast as the absorber cells. Thepositive phage clones were deduced by DNA sequencing. Eighteen positive phageclones were found out, whose specific binding force against human osteoblasts weremore than2times that of human gingival fibroblasts cells. The highest ratio was4.88.The purified phage were experiment vie immunofluorescence detection, whoseamino acid sequence was V L A V P W S E P GY L.The positive phage clones were found to be gathered on the surface of humanosteoblast after immunofluorescence detection, which were gathered inside cells andgave out green fluorescent. However, the human gingival fibroblasts can not gave outgreen fluorescent. Amino acid composition and their basic biochemical properties ofshort peptide （V L A V P W S E P G Y L）were analyzed.Conclusion: The specific peptide against human osteoblasts can be obtainedfrom a phage-display peptide library for use as a new research approach andexperimental basis of the biolization modification of the titanium surface.