中华预防医学杂志
中華預防醫學雜誌
중화예방의학잡지
CHINESE JOURNAL OF
2011年
1期
12-16
,共5页
杨焕森%胡忠义%刘忠华%王洁%沙巍%杨华
楊煥森%鬍忠義%劉忠華%王潔%沙巍%楊華
양환삼%호충의%류충화%왕길%사외%양화
分枝杆菌,结核%肽库%免疫测定%C肽
分枝桿菌,結覈%肽庫%免疫測定%C肽
분지간균,결핵%태고%면역측정%C태
Mycobacterium tuberculosis%Peptide library%Immunoassay%C-peptide
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.
目的 利用純化的結覈病患者血清IgG從噬菌體展示隨機7肽庫中篩選結覈特異性抗體結閤肽,為下一步開髮新的結覈病血清學檢測試劑提供思路.方法 以純化後結覈病患者血清IgG作為固相配基,按吸附、洗脫、擴增的生物淘洗(簡稱淘選)過程對噬菌體展示隨機7肽庫進行篩選,併于第2至3輪的篩選中加入純化後健康人血清IgG進行反篩,經過3輪淘選使噬菌體得到富集,分彆從滴度測定平闆上各箇方位挑取結覈病患者IgG和健康人IgG洗脫單噬菌體各20箇進行擴增純化,提取單鏈DNA作為模闆進行測序,間接ELISA法檢測不同單噬菌體與結覈病患者和健康人IgG的結閤情況,鑒定齣暘性剋隆.取收集的47份結覈病患者和37份有卡介苗接種史的健康人血清標本,採用phage-ELISA法對穫得的暘性單噬菌體進行初步驗證,併對其中24份篩選用血清標本的檢測結果進行分彆統計.結果 通過3輪淘選,能與靶分子特異性結閤的噬菌體得到明顯富集.單噬菌體測序分析共穫得12種共同序列.12種序列各取1箇剋隆擴增後進行間接ELISA檢測,結果錶明單噬菌體H12與結覈患者IgG具有較高的親和力(S/N≥2.1),確定為暘性剋隆.間接ELISA檢測髮現,單噬菌體H12對47份結覈病患者血清標本的檢齣值(0.105±0.010)明顯高于對37份健康人的檢齣值(0.070±0.005),t=2.912,P<0.0001.對其中篩選用12份結覈病患者和12份健康人血清標本的檢齣值分彆為0.144±0.016、0.052±0.004,差異同樣具有統計學意義(t=5.69,P<0.0001).結論 利用噬菌體展示隨機肽庫淘選穫得瞭結覈特異性抗體結閤肽,顯示齣與結覈病患者IgG較特異的結閤活性,為以多肽為基礎探索新的結覈病血清學診斷方法提供瞭依據.
목적 이용순화적결핵병환자혈청IgG종서균체전시수궤7태고중사선결핵특이성항체결합태,위하일보개발신적결핵병혈청학검측시제제공사로.방법 이순화후결핵병환자혈청IgG작위고상배기,안흡부、세탈、확증적생물도세(간칭도선)과정대서균체전시수궤7태고진행사선,병우제2지3륜적사선중가입순화후건강인혈청IgG진행반사,경과3륜도선사서균체득도부집,분별종적도측정평판상각개방위도취결핵병환자IgG화건강인IgG세탈단서균체각20개진행확증순화,제취단련DNA작위모판진행측서,간접ELISA법검측불동단서균체여결핵병환자화건강인IgG적결합정황,감정출양성극륭.취수집적47빈결핵병환자화37빈유잡개묘접충사적건강인혈청표본,채용phage-ELISA법대획득적양성단서균체진행초보험증,병대기중24빈사선용혈청표본적검측결과진행분별통계.결과 통과3륜도선,능여파분자특이성결합적서균체득도명현부집.단서균체측서분석공획득12충공동서렬.12충서렬각취1개극륭확증후진행간접ELISA검측,결과표명단서균체H12여결핵환자IgG구유교고적친화력(S/N≥2.1),학정위양성극륭.간접ELISA검측발현,단서균체H12대47빈결핵병환자혈청표본적검출치(0.105±0.010)명현고우대37빈건강인적검출치(0.070±0.005),t=2.912,P<0.0001.대기중사선용12빈결핵병환자화12빈건강인혈청표본적검출치분별위0.144±0.016、0.052±0.004,차이동양구유통계학의의(t=5.69,P<0.0001).결론 이용서균체전시수궤태고도선획득료결핵특이성항체결합태,현시출여결핵병환자IgG교특이적결합활성,위이다태위기출탐색신적결핵병혈청학진단방법제공료의거.
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 12 serum samples of tuberculosis patients and 12 samples of health individuals used for panning were 0. 144 ± 0. 016 and 0. 052 ± 0. 004, and the t value was 5. 69 (P <0. 0001). Conclusion By using phage-displayed random peptide libraries, we obtained the specific antibody binding peptides of tuberculosis,which showed specific binding activity with IgG of tuberculosis. It can provide a basis for the establishment of a new serological detection method of tuberculosis with peptide.
