| Objective To obtain the antigenic epitopes which can affinitive bind to theHCV(hepatitis c virus) polyclonal antibodies by using a phage 12-mer peptide libraryand to test the efficiency of converse biopannning by using negative serum.Methods 1)Purify the Ig of the sera from a patient who infected with HCVby using (NH4)2SO4 precipitation and a chromatography column constructed withDEAE Sephadex A-50. SDS-PAGE and ELISA(enzyme linked immunosorbent assay )were used to analysis the purified Ig. 2) Three rounds of biopanning were carried outby using the purified protein and the phage 12-mer peptide library. Conversebiopanning were also done at the same time. 3) ELISA were carried out to examinethe efficiency of the biopanning and the HCV specificity of the 26 phage cloneswhich were picked out randomly after the third round of biopanning. 4) Using westernblot to analyse the type of the epitopes which were got from the polyclonal phagesafter three round of biopanning and the positive monoclonal phages. 5) Purify thesingle chain templates of the positive phage clones to determine the sequence, whichwas inserted in the phage vector, and deduce the corresponding amino acid. 6) Locatethe same or similar amino acid on the 3-D model of HCV NS3 protein and/or analysethe antigenicity, hydrophobicity and solvent accessibility characters of the amino acidsequence displayed on the positive phage clones.Results 1) SDS-PAGE indicated that the purified protein was pure on theelectrophoresis level. The purified protein indicates a good affinity to HCV antigenand has a very high titer by ELISA. 2) The output/input value of the secondbiopanning was lower than the comparison one. The value of the comparativebiopanning is 5.0×10-5, while the value of the biopanning is 4.4×10-8. Theoutput/input value of the third biopanning is 30 times higher than that of the secondbiopanning. 3) Five of 26 clones can specific bind to HCV positive sera while notbind to HCV negative sera (p<0.05). The result of western blot indicates that therewere linear epitopes included in the polyclonal phages which were got after threeround of biopanning. 4) The sequences of the five positive phage clones weredetermined and there were two clones sharing the same sequence. Sequence analysisshows that: the peptide sequence YFQVGLESIPRP has a homologous one at aminoacids 1082-1093 of HCV protein and sequence MNALRPLSPWPT has a homologousone at amino acids 1954-1965 of HCV protein. 5) The consensus or similar aminoacids of peptide sequence YFQVGLESIPRP are located on the 3D model of HCVNS3. It shows that they are on the surface of the protein and join each other. Thehomologous sequences of the HCV protein show a good character as an antigen. Conclusion The converse biopanning by using negative sera can improve theefficiency of screen. After a series of examinations, the sequences MNALRPLSPWPT,YFQVGLESIPRP and YSQDRPASLTRS, MNKVPYYSINDS are likely to mimic theepitopes of HCV. It gives us a clue to improve the efficiency of immunologicaldiagnosis by using short peptide epitopes got from phage display library. |
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