鼻咽癌患者血清抗体相关抗原优势表位的筛选  

Screening of Dominant Epitopes of Antigens Related to Antibodies in the Sera of Patients with Nasopharyngeal Carcinoma

作　　者：张颖[1] 肖锡宾[1] 张昌卿[1] 李经略[1] 孙韻[1] 叶永照[1] 冯凯涛[1] 

机构地区：[1]中山大学肿瘤防治中心实验研究部,广东广州510060

出　　处：《癌症》2004年第9期999-1004,共6页Chinese Journal of Cancer

摘　　要：背景与目的:EB病毒与鼻咽癌(nasopharyngealcarcinoma,NPC)密切相关。可应用多种血清学方法检测鼻咽癌患者血清EBV抗体,本研究利用鼻咽癌患者血清中纯化的EB病毒相关多抗,从噬菌体展示的随机肽库中筛选相关的优势抗原表位,在表位水平为鼻咽癌患者血清抗体的检测寻找新的检测抗原。方法:以B95-8细胞EBV蛋白为抗原,从鼻咽癌患者血清中洗脱EB病毒相关多抗,对噬菌体展示的随机12肽库进行三轮生物淘洗。用夹心ELISA法和竞争抑制实验筛选阳性克隆,对这些阳性克隆进行测序,并将其展示的外源多肽与EB病毒蛋白的抗原性区域进行同源序列比对分析。结果:从第三轮的淘洗洗脱液中随机挑取64个噬菌体克隆,用夹心ELISA法筛选到25个阳性克隆,阳性率为39.06%,选取其中13个克隆进行竞争ELISA实验,有11个克隆出现抑制现象,抑制率在18.09%～65.94%之间。选取吸光度(A)值和抑制率均较高的5个克隆为阳性克隆,它们所展示的外源多肽序列分别为-A-T-S-H-L-H-V-R-L-P-W-T-(d15/d18)、-G-S-T-H-K-H-N-H-F-N-K-T-(d19)、-K-P-I-H-E-H-P-H-R-F-K-S-(e8)、-H-T-H-K-I-K-I-P-L-P-I-Q-(e23)。这些多肽序列分别与EB病毒膜蛋白(d15/d18)、EB病毒胸苷激酶(d19)、EB病毒主要壳蛋白(e8,e23)抗原区域的氨基酸存在序列相似性。BACKGROUND &OBJECTIVE: Epstein-Barr virus (EBV) is closely rela te d to nasopharyngeal carcinoma (NPC). Many kinds of methods can be used to examin e antibodies in NPC patients sera. This study was to screen the dominant epitope s from random peptide libraries (RPLs) displayed on phage using the EBV-related antibodies purified from the sera of NPC patients, and find new antigens at the epitope level. METHODS: The EBV-related antibodies were eluted from the sera o f NPC patients using B95-8 cell EBV proteins as antigen, and the phages from 12-mer RPLs were elutriated for 3 rounds with the antibodies.The positive clones were gained by sandwich ELISA, and competitive inhibition assay from the third e lution. The positive clones were sequenced, and the peptide coded by the inserte d DNA were blasted with the antigen region of EBV proteins. RESULTS: Sixty-four phage clones were randomly picked up from the third eluate,25 positive clones w ere picked up using sandwich ELISA assay, the positive percentage was 39.06%. T hirteen clones were picked up for competitive ELISA assay,11 clones showed inhib itory phenomena,the inhibitory rates were between 18.09%and 65.94%. Five posit ive clones with high absorbency value, and high inhibitory rates were selected o ut, the sequences of peptides displayed on these clones were-A-T-S-H-L-H-V-R-L-P-W-T-(d15, and d18),-G-S-T-H-K-H-N-H-F-N-K-T-(d19),-K-P-I-H-E-H-P-H-R-F-K-S-(e8),-H-T-H-K-I-K-I-P-L-P-I-Q-(e23). These peptide sequences showed similarity with the amino acid sequence s located in antigen regions of EBV integral membrane protein (d15, and d18), EB V thymidine kinase (d19), and EBV major capsid protein (e8, and e23). CONCLUSION : EBV-related epitopes could be obtained by screening the phages from RPLs with polyclonal antibodies purified from the sera of NPC patients, which may offer n ew methods of antigen preparation for sera diagnosis of NPC.

关 键 词：鼻咽肿瘤 抗原表位 随机多肽文库 多克隆抗体 

分 类 号：R739.63[医药卫生—肿瘤]