Research Of Peptidic Vector For Target Therapy Of Breast Cancer

Objective:To select tumor cell specific peptide vector that be internalized into MDA -MB-231cells and investigate the cell-type specificity and delivery efficiency and develop a targeting vector for tumor target therapy.Method:MDA-MB-231 cell,a human breast cancer cell line,was 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,then their targeting capacity to MDA-MB-231 cells were detected by coculturing them with different cell lines.DNA encoding CASPSGALRSC was ligated into plasmid pGEX-2T for fusion protein expression.The expression of fusion protein in BL21 (DE3)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:MDA-MB-231 cell specific peptide presenting phages were isolated from phage libraries,which showed higher affinity to MDA-MB-231 cell than other cell lines.Five peptide,including CASPSGALRSC(Pâ… ),CFPVPGHDLVC(Pâ…¡), CFSVPGHDIVC(Pâ…¢),CYTYPLGWHIC(Pâ…£)and CTPMSLSLSEC(Pâ…¤) with MDA-MB-231cell specificity were identified by DNA sequencing.Peptide without phage coat protein keep internalization character in MDA-MB-231 cell and showed juxtanuclear localization.The selected Peptide has higher affinity to MDA-MB-231 cells compared with controlled RGD-integrin.The recombinant plasmid vector encoding MDA-MB-231 specific peptide was successfully constructed and the purification of fusion protein 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 gene therapy.