【作者】 汤兆明；

【导师】 郭永建； 苏东辉；

【作者基本信息】 福建医科大学 ， 免疫学， 2005， 硕士

【摘要】 目的筛选噬菌体展示随机12 肽库中可以与HCV 抗体阳性的血清特异性结合的序列,在HCV 表达的蛋白分子上找到这个抗原表位, 分析其序列、结构特点与功能的关系。同时检验用阴性血清逆向筛选是否能提高筛选效率。从而探讨用噬菌体展示技术模拟HCV 抗原表位短肽,并用于对HCV 抗体检测的应用前景。方法1. 用饱和硫酸铵盐析法初提HCV 血清免疫球蛋白,再用DEAESehpadex A-50 进一步纯化。SDS-PAGE 鉴定纯化蛋白的纯度,ELISA 检测纯化蛋白的活性。2. 用纯化免疫球蛋白对噬菌体展示随机12 肽库进行3 轮亲和筛选并同时进行HCV 阴性血清逆向筛选。3. ELISA 方法检测各轮筛选的富集效果以及第三轮筛选后随机挑选的26 个噬菌体单克隆和HCV 阴性和阳性血清反应的特异性。4. Western blot 检测各阳性噬菌体单克隆展示多肽表位的类型以及筛选后所得噬菌体多克隆所展示多肽的抗原类型。5. 提取特异性噬菌体克隆单链DNA 并测定插入的外源序列。6. 利用生物信息学软件呈现噬菌体展示多肽的三维结构;分析表位多肽的抗原相关性质。结果1) 经过两步纯化后收集到免疫球蛋白的洗脱混合液,经SDS-PAGE电泳后未见杂带,达到电泳纯。纯化免疫球蛋白与HCV抗原ELISA反应结果为强阳性。2) 在第二轮筛选中经HCV阴性血清逆向筛选后的产出较对照明显偏低。对照孔产出/投入为5.0×10-5,经逆向吸收的筛选孔产出/投入为4.4×10-8。第三轮的产出/投入值较第二轮提高30多倍。3) ELISA检测发现在26个噬菌体单克隆中有5个克隆能与多份HCV阳性血清结合而不与阴性血清反应,显示较好的特异性(P<0.05)。Western blot显示第三轮筛选收集的多克隆噬菌体中存在HCV线性表位。4) 经DNA测序发现5个克隆有2个克隆的DNA序列是完全相同的,即5个克隆包含了4种不同的抗原表位;同源性分析显示YFQVGLESIPRP 与HCV蛋白分子1082～1093位的氨基酸同源性较高,而MNALRPLSPWPT与1954～1965位氨基酸同源性较高。5)与筛选表位序列YFQVGLESIPRP相同或相似的氨基酸在蛋白三维结构上紧密相连,形成抗原应有的构象。两个表位在HCV蛋白上的同源区表现出较好的抗原性质。更多还原

【Abstract】 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.更多还原