Expression Of P-glycoprotein Extracellular Fragment And Biospanning Of Its Specific Peptide

High expression of P-glycoprotein is mainly responsible for the multidrug resistance in human bladder cancer. The key to targeted treatment of tumor cells is to find specific targeted vector. Compared to antibody or antibody fragment, peptide vectors have the characters such as smaller molecular weight, good tissue penetrating ability, no induction of immune response, shorter half life, cleaning in time etc. So, the biospanning of peptide specific to P-glycoprotein is the premise for the targeted treatment of MDR in human bladder cancer, and it will certainly accelerate the progress both in the basic and clinical studies on MDR.In order to analysis structure and function in P-glycoprotein, to explore its surface properts and select P-glycoprotein extracellular fragment with high hydrophilicity and antigenicity expressed in E.Coli, based on the relative methodology, the second structure and surface properts of P-glycoprotein such as physics and chemical characters, hydrophilicity, antigenicity etc was predicted with many methods. Many distinct antigenic epitopes in P-glycoprotein were identified; According to prediction results of transmembrane region and hydrophilicity, The first extracellular fragment (82aa-115aa) of P-glycoprotein which including monoclonal antibody MRK16 epitope was selected as the targeted protein to be expressed; If used it to immune animals or biospan libraries, antibody or specific peptide would be identified. By designing PCR primer, the cDNA encoding for the extracellular fragment was amplified corresponding to the sequence in pHAMDRl/A plasmid which including the full length of mdrl cDNA. The product was cloned into pGEX-2T vector andtransfered into DH5a, expressed under optimum condition, GST-Pgp fused protein with high expression and hydrophilicity was acquired, its molecular weight is about 30 kD, and it was identified and purified. Using the fused protein to biospan in phage radom 12 peptide library, peptide specific binding to P-glycoprotein was acquired after 4 runs of biospanning. The inserted sequence of the peptide as NDGLLFTWQPSP was identified by sequencing analyses. Immunocytochemical staining in BIU-87/ADM and BIU-87 cells was done with positive phage clones, the results revealed that the binding only occurred in MDR cells, not in sensitive cells. It indicated that this kind of binding exerts its affinity and specificity to MDR cells.The predicted second structure and surface properts of P-glycoprotein can provide a base for studies on structure and function of P-glycoprotein, construction of its mutation or new form. The purified P-glycoprotein and assays established in this project facilitate the research in MDR. Also, the obtained peptide specific to P-glycoprotein place a experiment base for the diagnosis and targeted treatment of MDR in tumor cells .