鼻咽癌患者血清抗体相关抗原优势表位的筛选

背景与目的：EB病毒与鼻咽癌(nasopharyngeal carcinoma,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％之问。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一1-K-I-P-LP-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。结论：利用鼻咽癌患者血清中的特异多抗可以从噬菌体展示的随机肽库中筛选到EB病毒相关抗原表位。

Screening of dominant epitopes of antigens related to antibodies in the sera of patients with nasopharyngeal carcinoma

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.