Identification And Screening Of Mimic Epitope Of MUC1

Mucins (MUC1) are high molecular weight glycoproteins secreted by many epithelial cells such as bladder , breast, ovary and pancreatic carcinomas.MUC1 is of interest and a target for tumour immunotherapy because: (1) there is up to a 100-fold increase in the amount of mucin present on cancer cells compared to normal cells. (2) it has a ubiquitous, rather than focal cellular distribution and (3) it has altered glycosylation, revealing peptide epitopes not easily identified in normal mucins. It was indicated that most of the immunogenicity, resided in a repeated increase in the amount of mucin present on cancer VNTR(variable number of tandem repeats,VNTRs) 20 amino acid peptide domain in the extracellular portion of the molecule. MUC1 can stimulate T cells in a non-MHC-restricted manner by recognized by T cell receptor . In addition to T cell immunity, a B-cell immune response to epitopes of MUC1 to produce antibody also has been demonstrated. At present ,many vaccines based on MUC1 antigen have been designed in tumor therapy research ,some have been used in clinical tumor therapy . In order to analyze the structure and function of MUC1 to explore its surface properts, the secondary structure and surface properts of MUC1 such as physics and chemical characters, hydrophilicity, antigenicity and so on was predicted with various methods. Many distinct antigenic epitopes in MUC1 were identified by computation; According to prediction results of antigenicity, the extracellular fragment of MUC1 which include VNTRs was found containing antigenic epitope such as 140aa-156aa, 159aa-174aa, 179aa-196aa; BC2 Ab specific to MUC1 raised from mouse was purified with Ion Exchange Chromatography method. the purified BC2 Ab was used to biospan in phage radom 7 peptide library and mimic epitopes were acquired after 3 rounds of biospanning. The inserted sequences of the peptides as TAPDLRP(x19),SAPDLRP(x9),AAPDSRP(x1)and LAPDFRP (x1)were identified by sequencing analyses and the motif was identified as XAPDXRP .The inhibitory assay showed that the 4 mimic epitope peptides displaying on the phage surface could effectivaly inhibit the combination of antibody with antigen and the inhibitory rates of eath mimic epitope were 50% higher than the control, which indicated that the identified peptides could simulate the epitope of MUC1 . In order to evaluate the immunological characteristics of these mimic epitopes, mice were immunized with peptide TAPDLRP. Ab specific to MUC1 could be detected in the mice serum following 3 times of immunization with TAPDLRP. Western Blot was made by using the purified serum of biggest OD450 value .The results showed that the purified serum can recognize MUC1 protein ..proliferation assays of T-cells from spleen of Balb/C inoculated with the positive phages showed that T-cells were markely proliferated in absence of adjuvant during immunization , which demenstrated that phage might be a proper vector for the immunization. These results revealed that MUC1 mimic peptide displaying on the surface of phage had good immunological properties to produce humoral and cellular immune responses.The predicted secondary structure and surface properts of MUC1 can provide a basic clues for studies on structure and function of MUC1, construction of its mutation or new form. The obtained mimic epitopes specific to MUC1 can provide primary experiment data for the development of candidate vaccine for epitheliomas.