摘要
目的 利用纯化的结核病患者血清IgG从噬菌体展示随机7肽库中筛选结核特异性抗体结合肽,为下一步开发新的结核病血清学检测试剂提供思路.方法 以纯化后结核病患者血清IgG作为固相配基,按吸附、洗脱、扩增的生物淘洗（简称淘选）过程对噬菌体展示随机7肽库进行筛选,并于第2至3轮的筛选中加入纯化后健康人血清IgG进行反筛,经过3轮淘选使噬菌体得到富集,分别从滴度测定平板上各个方位挑取结核病患者IgG和健康人IgG洗脱单噬菌体各20个进行扩增纯化,提取单链DNA作为模板进行测序,间接ELISA法检测不同单噬菌体与结核病患者和健康人IgG的结合情况,鉴定出阳性克隆.取收集的47份结核病患者和37份有卡介苗接种史的健康人血清标本,采用phage-ELISA法对获得的阳性单噬菌体进行初步验证,并对其中24份筛选用血清标本的检测结果进行分别统计.结果 通过3轮淘选,能与靶分子特异性结合的噬菌体得到明显富集.单噬菌体测序分析共获得12种共同序列.12种序列各取1个克隆扩增后进行间接ELISA检测,结果表明单噬菌体H12与结核患者IgG具有较高的亲和力（S/N≥2.1）,确定为阳性克隆.间接ELISA检测发现,单噬菌体H12对47份结核病患者血清标本的检出值（0.105±0.010）明显高于对37份健康人的检出值（0.070±0.005）,t=2.912,P〈0.0001.对其中筛选用12份结核病患者和12份健康人血清标本的检出值分别为0.144±0.016、0.052±0.004,差异同样具有统计学意义（t=5.69,P〈0.0001）.结论 利用噬菌体展示随机肽库淘选获得了结核特异性抗体结合肽,显示出与结核病患者IgG较特异的结合活性,为以多肽为基础探索新的结核病血清学诊断方法提供了依据.
Objective To screen the specific antibody binding peptides of tuberculosis from phagedisplayed random phage display（Ph. D.）-7 peptide libraries with purified IgG from tuberculosis serum ,and to provide the basis for the development of serological detection reagent of tuberculosis. Methods Purified IgG of tuberculosis serum was used as solid ligand to screen the binding peptide from the Ph. D.-7 peptide library,according to the biopanning process of absorption, elution and amplification. Purified IgG of health people serum was used as the molecule of counter selection during the second and third selection. Phages were enriched after 3 rounds of screening, then 20 single phages separately eluted by IgG of tuberculosis and health people were randomly selected on each direction of the determination plates and amplified. The single chains DNA were extracted as template for sequencing. The combination abilities of selected clones to IgG of tuberculosis and health people were tested by indirect enzyme linked immunosorbent assay （ELISA） ,and the positive clone was identified. Serum samples, from 47 patients with tuberculosis and 37 healthy people vaccinated with BCG, were verified by positive phage clones using phage-ELISA. The verifying results of 24 serum samples used for panning were separately analyzed statistically. Results After 3 rounds of panning, remarkable enrichment of phages that could specifically bind with target molecules were observed. Single phages were randomly selected for sequencing analysis and 12 sequences were obtained.12 phage clones with different sequences were amplified and detected with indirect ELISA and single phage H12 showed higher affinity with IgG of tuberculosis（S/N ≥2. 1）and was identified as the positive clone. It was found that,in indirect ELISA with singe Phage H12,the A450 value of tuberculosis patients （0. 105 ±0. 010） was significantly higher than that of healthy individuals （0. 070 ± 0. 005）, and the t value was 2.912 （P〈 0. 0001）. The A450 value of
出处
《中华预防医学杂志》
CAS
CSCD
北大核心
2011年第1期12-16,共5页
Chinese Journal of Preventive Medicine
基金
基金项目：国家科技重大专项（2008ZX100034305）
上海市自然科学基金项目（09ZR1425800）
关键词
分枝杆菌
结核
肽库
免疫测定
C肽
Mycobacterium tuberculosis
Peptide library
Immunoassay
C-peptide
