The Protein Transduction Peptide Selected From Phage Peptide Library To Target The Original And Bone Metastasis Cells Of Breast Cancer Cell Line-MDA-MB-231

OBJECTIVE:To estabilish a animal model of bone metastasis of breast carcinoma and select tumor cell specific peptide vector that be internalized into MDA -MB-231 cells/its bone metastasis cell and investigate the cell-type specificity and delivery efficiency in vitro.METHOD:To develop a targeting vector for target therapy of breast cancer.We establish animal model of breast cancer bone metastasis using human breast cancer cell lines MDA-MB-231 transfected with GFP via left ventricle of heart.After 45 days of injection,all mice were detected by X-ray/PET-CT to determine whether bone metastasis happened in mouse.The cells from bone metastasis were observed the GFP expression under inverted fluorescence microscope,then cultured for further study.PC 89(11aa),a phage display library of random peptides,was applifed until its tite reaching 10¹¹TU.MDA-MB-231 cells and bone metastasis cells were cocultured with pC89(11aa)phage display library of random peptides.In multiple independent peptide-presenting phage screening trials,subtilisin was used as a proteinese inhibitor to inactivate extra-cellular phages.The internalized phages were collected by cell lysising and amplified in E.Coli XLI-Blue.Through five rounds of selection,the peptide-presenting phages which could be internalized in MDA-MB-231 cells were isolated.The internalization capacity of peptide-presenting phages isolated from MDA-MB-231 cells was following compared with RGD-integrin binding phage by coculturing them with other human tumor cell lines and normal cells.The nucleotide sequences of isolated peptide-presenting phages were then determined by DNA sequencing.Peptide without phage coat protein were synthesized labeled with FITC, and,then their targeting capacity to MDA-MB-231 cells were detected by coculturing them with different cell lines.DNA encoding specific peptide to MDA-MB-231 were ligated into plasmid pGEX-2T for fusion protein expression.The expression of fusion protein was identified by SDS-PAGE and western-blotting analysis.The delivery efficiency of the peptide was tested by anti-GST antibody in different breast cancer cell lines.Result:A animal model of bone metastasis of breast Cancer was established using human breast cancer cell lines MDA-MB-231 transfected with GFP via left ventricle of heart.62.5%mice were found the metastasis in bone,lung and other tissues.The specific peptide presenting phages were isolated from phage libraries,which showed higher affinity to MDA-MB-231 cells and/or bone matastasis cells than other cell lines. Five peptide,including CASPSGALRSC(Pâ… ),CFPVPGHDLVC(Pâ…¡), CFSVPGHDIVC(Pâ…¢),CYTYPLGWHIC(Pâ…£)and CTPMSLSLSEC(Pâ…¤)were identified by DNA sequencing.Compared with controlled RGD-integrin Phage,The phage,encoding Pâ… sequence,show high affility in both MDA-MB-231 cell and bone matastasis cell,but Pâ…¡phage(encoding Pâ…¡sequence)can be higher internalized in bone matastasis cells than MDA-MB-231 cells.Peptides(Pâ… ,Pâ…¡)without phage coat protein keep internalization character and showed juxtanuclear localization.No affility from five peptides were detected in all other tumor cells and normal cells that we used in our study.The recombinant plasmid vector(PGEX-2T-Pâ… ,PGEX-2T-Pâ…¡)encoding MDA-MB-231 specific peptide(Pâ… ,Pâ…¡)were successfully constructed,both fusion proteins(Pâ… -GST,Pâ…¡-GST)were applified in E coil BL21 and purified with GST.affinity chrornatographthe.The ratio of purification reached to 85%.Immunoflenscense microscopy showed that the peptide mediated the intracellular transduction of fusion protein targetingly in MDA-MB-231 cells.Conclusion:In conclusion,we established a method to seek novel tumor specific peptide,which may also be a practical way to promote the research in the area of new cell receptor or antigen.Capacity of Pâ… to deliver molecules into target cells confirms its therapeutic potent in individualization targeting therapy.