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Objective:To analysis the Mimotopes of the peptide mimics to PGN using online softwares.Methods: Mimotopes of PGN were screened from 12-mers linear phage display peptide library by using anti-PGN McAb and the antigenicity of selected clones was identified by ELISA.The B cell epitopes,T cell epitopes of ′GRWxHxVxWAGL′ were estimated by DNAstar and online softwares.Results: 16 phage clones that bound with anti-PGN McAb were screened from 12-mers linear phage display peptide library.Among these positive clones,phage clones No.39 shared the conserved sequence:WxHx……AGL found in previous clone No.31(ATWxHxLxSAGL),which provoked an effective protective immunity against infection with S.aureus.To enhance the stability of the conformation as well as adding biotin on the N-ter-minal as a tag,the sequences ′39′ were redesigned and synthesized by adding S(serine)A(alanine) and GG(glycine)on the C-terminal of origin sequence(named SP39).Next,we estimated or predicted antigenic epitopes,T cell epitopes and scores binding to MHC of these peptides by using DNASTAR and online softwares(http://bio.dfci.harvard.edu/Tools/antigenic.pl,www.syfpeithi.de,http://www.darrenflower.info/mhcpred),indicating that SP39 contains sites bound both mice and human MHC.The sequences ′WxHxVxW-′ may be antigenic epitope as SP39,which contains a T cell epitope.Our results showed that both SP39 could bind to both anti-PGN McAb and a polyclonal antibody against S.aureus.Moreover PGN could inhibit the binding of SP39 to the anti-PGN McAb.These data indicated that SP39 mimic to epitopes on PGN.Conclusion: SP39(GRWxHxVxWAGLAGGS) probably display the mimotopes of PGN.
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Objective To screen and identify the phage-display random 7 amino acid peptide specific to the systemic lupus erythematosus(SLE) and analyze its practical significance. Methods Using the phage random 7 peptide library screening, the SLE specific phage clones are obtained after binding with the mixture of sera from 30 SLE patients and 30 normal controls as ligand respectively. Then the Dot-ELISA is used to identify the SLE specific phage clones reactive to sera of the SLE patients and normal controls individually. Finally the identified phage-display random 7 amino acid peptides are sequenced and it's homology with the antigenic epitope of human being and other are also analyzed. Results Total 12 of the phage-display random 7 amino acid peptide are obtained by phage peptide library screening and the Dot-ELISA identification. Sequence analysis shows that the identified phage-display random 7 amino acid peptide epitope have homology with E. coli, Salmonella and human immunodeficiency virus, but not with that of human being. Conclusion SLE-specific peptides screened by phage random peptide library maybe used to diagnosis the SLE. Meanwhile, the antibodies in SLE patients which are combined with the Pathogen epitope, suggest that SLE maybe relate to pathogen infection.
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Objective: To establish a lung cancer phage-peptide library and to screen for biomarkers for early detection of lung cancer. Methods: A phage display library was constructed using 30 lung-cancer tissue samples from Changhai Hospital. Protein-A/G agarose was used to enrich IgG from control sera as well as lung cancer sera. Five biopannings were carried out for enrichment of lung cancer-specific phage clones. Five hundred phage clones were randomly selected,those with D>2.0(lung cancer plasma pool v. s. control plasma pool) were selected for DNA sequencing and protein prediction. Results: (1) The recombination rate of the phage library was 60%,with the average phage titer being 3.0 × 106 pfu and a volume being 9.0 × 106 pfu. (2) Of 19 phage colonies selected by ELISA and sequenced,9 were cancer-related genes,8 with unknown function, and 2 were not related to cancer. Conclusion: The screened genes in the phage colonies might serve as biomarkers for the early diagnosis of lung cancer.
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Objective:To identify monoclonal antibody McAb2 recognizing epitope of Fba of GAS.Methods:The overlapped peptides were synthesized and their abilities to bind McAb2 were detected by dot-ELISA.The predominance amino acids specific for McAb2 were screened using phage 7 peptide library.Results:The result by dot-ELISA analysis demonstrated that the synthetized peptide,amino-acid residues 100-112~(th),could bind McAb2 with high affinity.The predominance amino acids specific for McAb2 were ITPDL,which was located in 100-110~(th)aa of Fba by panning with phage 7 peptide library.Conclusion:The domain and the predominance amino acids of Fba recognized by McAb2 is determined.The results would contribute to the research of the role of Fba on the pathogenic mechanism of GAS,the identification of function of McAb2,and the development of epitope-peptide vaccine.
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Objective To identify epitope of neutrophil-activating protein(NAP) of Helicobacter pylori(Hp).Methods Using the mouse monoclonal antibodies against NAP as selective molecular and immunoscrcening phage-display random 7-peptides library.The positive clones were sequenced and analyzed.Phage clones were chosen to immunize mice and to evaluate the potential of phagotopes as effective vaccines.Results One mimotope(FAHLATQ)showed a good match with the NAP at 140-143 AA(AHLA)and the serum of mice induced by the phage clone clearly recognized NAP.Conclusion This study suggests thatthe antigenic epitope could be mapped through screening the phage-display peptide library with monoclonalantibody and a mimotopo of NAP providing an ahernative approach for the diagnosis and development of avaccine for Hp.
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BACKGROUND: Hepatitis C virus(HCV), a family of Flaviviridae, has a host cell-derived envelope containing a positive-stranded RNA genome, and has been known as the maj or etiological agent for chronic hepatitis, hepatic cirrhosis, and hepatocellular carcinoma. There remains a need to dissect a molecular mechanism of pathogenesis for the development of therapeutic and effective preventive measure for HCV. Identification of cellular receptor is of central importance not only to understand the viral pathogenesis, but also to exploit strategies for prevention of HCV. This study was aimed at identifying peptide mimotopes inhibiting the binding of E2 protein of HCV to MOLT-4 cell . METHODS: In this study, phage peptide library displaying a random peptides consisting of 7 or 12 random peptides was employed in order to pan against E2 protein. Free HCV particles were separated from the immune complex forms by immunoprecipitation using anti-human IgG antibody, and used for HCV-capture ELISA. To identify the peptides inhibiting E2-binding to MOLT-4 cells, E2 protein was subj ect to bind to MOLT-4 cells under the competition with phage peptides. RESULTS: Several phage peptides were selected for their specific binding to E2 protein, which showed the conserved sequence of SHFWRAP from 3 different peptide sequences. They were also able to recognize the HCV particles in the sera of HCV patient s captured by monoclonal antibody against E2 protein. Two of them, showing peptide sequence of HLGPWMSHWFQR and WAPPLERSSLFY respectively, were revealed to inhibit the binding of E2 protein to MOLT-4 cell efficiently in dose dependent mode. However, few membrane-associated receptor candidates were seen using Fasta3 programe for homology search with these peptides. CONCLUSION: Phage peptides containing HLGPWMSHWFQR and WAPPLERSSLFY respectively, showed the inhibition of E2-binding to MOLT-4 cells. However, they did not reveal any homologues to cellular receptors from GenBank database. In further study, cellular receptor could be identified through the screening of cDNA library from MOLT-4 or hepatocytes using antibodies against these peptide mimotopes.
Subject(s)
Humans , Antibodies , Antigen-Antibody Complex , Bacteriophages , Carcinoma, Hepatocellular , Conserved Sequence , Databases, Nucleic Acid , Enzyme-Linked Immunosorbent Assay , Flaviviridae , Gene Library , Genome , Hepacivirus , Hepatitis C , Hepatitis , Hepatitis, Chronic , Hepatocytes , Immunoglobulin G , Immunoprecipitation , Liver Cirrhosis , Mass Screening , Peptide Library , Peptides , RNAABSTRACT
To screen HCV core mimotopes,HCV core monoclonal antibody was used as immobilized molecule,and a 12 mer phage peptide library was biopanned and positive clones were selected by enzyme linked immunoadsorbent assay(ELISA), competitive inhibition assay, and DNA sequencing. 10 positive clones were chosen for DNA sequencing. From the experiment and sequencing comparison results,one epitope was confirmed as a mimotope of HCV core protein. The study might provide a new approach for HCV therapy and vaccine development.