中华微生物学和免疫学杂志
中華微生物學和免疫學雜誌
중화미생물학화면역학잡지
CHINESE JOURNAL OF MICROBIOLOGY AND IMMUNOLOGY
2011年
8期
713-717
,共5页
陆晔%林成招%胡衡%梅建%武洁%沈鑫
陸曄%林成招%鬍衡%梅建%武潔%瀋鑫
륙엽%림성초%호형%매건%무길%침흠
结核分枝杆菌%异烟肼耐药%二维液相色谱-质谱技术%亚细胞蛋白质组
結覈分枝桿菌%異煙肼耐藥%二維液相色譜-質譜技術%亞細胞蛋白質組
결핵분지간균%이연정내약%이유액상색보-질보기술%아세포단백질조
Mycobacterium tuberculosis%Isoniazid-resistant%2-dimensional liquid chromatography%Sub-cellular proteome
目的 研究结核分枝杆菌异烟肼耐药株和敏感株亚细胞蛋白质组差异，鉴定菌体细胞壁蛋白和细胞膜蛋白中与异烟肼耐药相关的蛋白，并初步探讨其在临床检验中的应用价值。方法 应用密度梯度离心法分离5株异烟肼耐药株和5株敏感株的细胞壁和细胞膜蛋白，利用二维液相色谱分离技术进一步获得耐药株和敏感株的亚细胞蛋白表达差异图谱。运用基质辅助激光解析/电离飞行时间质谱技术对获得的1280个细胞壁和细胞膜组分进行质谱鉴定，并运用DAVID数据库中GO注释功能对鉴定到的蛋白进行细胞成分和生物过程的注释和分类。运用NASF技术对蛋白表达进行定量，报告阈值为1.5。选取5个在耐药株中表达上调、2个在敏感株中表达上调的蛋白分别与菌株来源患者的血清进行ELISA检测，检测上述组分与血清样品发生免疫应答的强度，组间比较采用t检验。结果 共鉴定到347个蛋白。蛋白定位分析表明58％蛋白定位于细胞膜或跨膜。蛋白功能聚类分析表明31％蛋白参与三羧酸循环，26％、15％蛋白分别参与脂类、脂肪酸生物合成和代谢，28％蛋白参与脂质体的生物合成、代谢、转运和定位。其中琥珀酰氯化胆碱合酶、单加氧酶、假想蛋白Rv2255c、烟碱核苷二甲基联苯酰磷酸核酮糖激酶、膜磷脂胞嘧啶转移酶在耐药株中表达上调，含有上述蛋白的组分与感染耐药株患者的血清免疫学反应的吸光度（A450值）明显高于与感染敏感株患者的血清免疫学反应强度，差异有统计学意义(t值分别为0.028、0.044、0.066、0.064、0.083，P均＜0.01)。Rv2002蛋白A链、Rv2632c蛋白A链在敏感株中表达上调，含有上述蛋白的组分与感染敏感株患者的血清免疫学反应的A450值明显高于与感染耐药株患者的血清免疫学反应强度，差异有统计学意义（t值分别为0.053、0.073，P均＜0.05）。结论 运用密度梯度离心法和二维液相色谱-质谱技术能在亚细胞水平上富集并鉴定结核分枝杆菌异烟肼耐药株和敏感株中表达有差异的蛋白。有助于寻找异烟肼耐药株感染相关的抗原蛋白，为进一步探讨结核分枝杆菌耐异烟肼机制、研究耐药株-宿主相互作用提供了有价值的线索。
目的 研究結覈分枝桿菌異煙肼耐藥株和敏感株亞細胞蛋白質組差異，鑒定菌體細胞壁蛋白和細胞膜蛋白中與異煙肼耐藥相關的蛋白，併初步探討其在臨床檢驗中的應用價值。方法 應用密度梯度離心法分離5株異煙肼耐藥株和5株敏感株的細胞壁和細胞膜蛋白，利用二維液相色譜分離技術進一步穫得耐藥株和敏感株的亞細胞蛋白錶達差異圖譜。運用基質輔助激光解析/電離飛行時間質譜技術對穫得的1280箇細胞壁和細胞膜組分進行質譜鑒定，併運用DAVID數據庫中GO註釋功能對鑒定到的蛋白進行細胞成分和生物過程的註釋和分類。運用NASF技術對蛋白錶達進行定量，報告閾值為1.5。選取5箇在耐藥株中錶達上調、2箇在敏感株中錶達上調的蛋白分彆與菌株來源患者的血清進行ELISA檢測，檢測上述組分與血清樣品髮生免疫應答的彊度，組間比較採用t檢驗。結果 共鑒定到347箇蛋白。蛋白定位分析錶明58％蛋白定位于細胞膜或跨膜。蛋白功能聚類分析錶明31％蛋白參與三羧痠循環，26％、15％蛋白分彆參與脂類、脂肪痠生物閤成和代謝，28％蛋白參與脂質體的生物閤成、代謝、轉運和定位。其中琥珀酰氯化膽堿閤酶、單加氧酶、假想蛋白Rv2255c、煙堿覈苷二甲基聯苯酰燐痠覈酮糖激酶、膜燐脂胞嘧啶轉移酶在耐藥株中錶達上調，含有上述蛋白的組分與感染耐藥株患者的血清免疫學反應的吸光度（A450值）明顯高于與感染敏感株患者的血清免疫學反應彊度，差異有統計學意義(t值分彆為0.028、0.044、0.066、0.064、0.083，P均＜0.01)。Rv2002蛋白A鏈、Rv2632c蛋白A鏈在敏感株中錶達上調，含有上述蛋白的組分與感染敏感株患者的血清免疫學反應的A450值明顯高于與感染耐藥株患者的血清免疫學反應彊度，差異有統計學意義（t值分彆為0.053、0.073，P均＜0.05）。結論 運用密度梯度離心法和二維液相色譜-質譜技術能在亞細胞水平上富集併鑒定結覈分枝桿菌異煙肼耐藥株和敏感株中錶達有差異的蛋白。有助于尋找異煙肼耐藥株感染相關的抗原蛋白，為進一步探討結覈分枝桿菌耐異煙肼機製、研究耐藥株-宿主相互作用提供瞭有價值的線索。
목적 연구결핵분지간균이연정내약주화민감주아세포단백질조차이，감정균체세포벽단백화세포막단백중여이연정내약상관적단백，병초보탐토기재림상검험중적응용개치。방법 응용밀도제도리심법분리5주이연정내약주화5주민감주적세포벽화세포막단백，이용이유액상색보분리기술진일보획득내약주화민감주적아세포단백표체차이도보。운용기질보조격광해석/전리비행시간질보기술대획득적1280개세포벽화세포막조분진행질보감정，병운용DAVID수거고중GO주석공능대감정도적단백진행세포성분화생물과정적주석화분류。운용NASF기술대단백표체진행정량，보고역치위1.5。선취5개재내약주중표체상조、2개재민감주중표체상조적단백분별여균주래원환자적혈청진행ELISA검측，검측상술조분여혈청양품발생면역응답적강도，조간비교채용t검험。결과 공감정도347개단백。단백정위분석표명58％단백정위우세포막혹과막。단백공능취류분석표명31％단백삼여삼최산순배，26％、15％단백분별삼여지류、지방산생물합성화대사，28％단백삼여지질체적생물합성、대사、전운화정위。기중호박선록화담감합매、단가양매、가상단백Rv2255c、연감핵감이갑기련분선린산핵동당격매、막린지포밀정전이매재내약주중표체상조，함유상술단백적조분여감염내약주환자적혈청면역학반응적흡광도（A450치）명현고우여감염민감주환자적혈청면역학반응강도，차이유통계학의의(t치분별위0.028、0.044、0.066、0.064、0.083，P균＜0.01)。Rv2002단백A련、Rv2632c단백A련재민감주중표체상조，함유상술단백적조분여감염민감주환자적혈청면역학반응적A450치명현고우여감염내약주환자적혈청면역학반응강도，차이유통계학의의（t치분별위0.053、0.073，P균＜0.05）。결론 운용밀도제도리심법화이유액상색보-질보기술능재아세포수평상부집병감정결핵분지간균이연정내약주화민감주중표체유차이적단백。유조우심조이연정내약주감염상관적항원단백，위진일보탐토결핵분지간균내이연정궤제、연구내약주-숙주상호작용제공료유개치적선색。
Objective To compare the sub-cellular proteome of isoniazid ( INH)-resistant Mycobacterium tuberculosis (MTB) with that of sensitive strains for identifying of unique proteins of these strains and discussing their preliminary application in clinical diagnosis. Methods Proteins of cell wall and membrane of 5 INH-resistant strains and 5 INH-sensitive strains were extracted by density gradient centrifugation.The extracts were subsequently analyzed using weak cation exchange (WCX) liquid chromatography ( LC )followed reverse phase (RP) liquid chromatography to compare the sub-cellular protein patterns. A total of 1280 fractions were collected and identified by matrix assisted laser desorption ionization-time of flight-tandem mass spectrometry (MALDI-TOF MS/MS). The Database for Annotation, Visualization and Integrated Discovery (DAVID) was used for cell component and biological process analysis. Normalized Spectral Abundance Factors (NASF) was used for semi-quantity of protein expression. 5 proteins significantly up-regulated in INH-resistant strains and 2 proteins significantly up-regulated in INH-sensitive strains were selected for ELISA analysis with autologous sera respectively. Results A total of 347 proteins were identified. Cell component analysis showed that 58％ proteins were cells well or membrane proteins. Biological process analysis showed that 31％ proteins involved in carboxylic/monocarboxylic acid biosynthetic and metabolic process, 26％ and 15％ proteins involved in organic acid or fatty acid biosynthetic and metabolic process,while 28％ proteins involved in lipid biosynthetic , metabolic, transport and localization process. O-succinylbenzoate synthase, monooxygenase, hypothetical protein Rv2255c, nicotinate-nucleotide--dimethylbenzimidazole phosphoribosyltransferase and membrane phosphatidate cytidylyltransferase cdsA were up-regulated in INH-resistant strains and fractions contained these proteins could elicit specific antibody response with autologous sera. The A450 was higher than that with INH-sensitive sera. The differences between the INH-resistant sera and the INH-sensitive sera were significant ( t = 0.028, 0.044, 0.066, 0.064, 0.083, all P＜0.01 ). Chain A of Rv2002 Gene Product and Chain A of Crystal Structure Of Rv2632c were up-regulated in INH-sensitive strains and fractions contained these proteins could elicit specific antibody response with autologous sera. The A450 was higher than that with INH-resistant sera. The differences between the INH-sensitive sera and the INH-resistant sera were significant (t=0.053, 0.073, both P＜0.05). Conclusion The combination of density gradient centrifugation and 2D-LC MS/MS technology is useful in enrichment and identification of differential expressed proteins between INH-resistant and INH-sensitive strains at sub-cellular level. It is useful in finding antigens associated with INH-resistant MTB infection, which may prove useful for further study in the mechanism of INH resistant, as well as interaction between MTB and host.
