CAJ | 학술논문

目的联合应用二维液相色谱(two-dimensional liquid phase fractionation,PF2D)及基质辅助激光解吸电离飞行时间串联质谱(matrix-assisted laser desorption ionization time-of-flight/time-of-flight mass spectrometry,MALDI-TOF/TOF-MS)技术,筛选多种牙龈卟啉单胞菌(Porphyromonas gingivalis,Pg)共同外膜蛋白(outer membrane protein,OMP),为针对多种Pg设计具有交叉保护作用的疫苗提供候选靶抗原.方法超高速离心法提取Pg301、PgATCC33277和PgW83菌株OMP,应用PF2D目标蛋白快速分离系统分离OMP,对比分析得到共同OMP,用MALDI-TOF/TOF-MS串联质谱结合数据库搜索,确定蛋白质一级结构.结果第一维色谱聚焦分离,3株Pg共获得99个蛋白质样本;选定OMP组分B7,进行第二维反相色谱分离,3株Pg共确定了8个共同的蛋白色谱峰,运用MALDI-TOF/TOF-MS对蛋白样品进行鉴定,结果确定了其中一个为已知的蛋白精氨酸-牙龈素A.结论 PF2D分离系统分辨率高、重现性好,结合MALDI-TOF/TOF-MS串联质谱技术,可用于鉴定Pg的共同OMP.
목적 연합응용이유액상색보(two-dimensional liquid phase fractionation,PF2D)급기질보조격광해흡전리비행시간천련질보(matrix-assisted laser desorption ionization time-of-flight/time-of-flight mass spectrometry,MALDI-TOF/TOF-MS)기술,사선다충아간계람단포균(Porphyromonas gingivalis,Pg)공동외막단백(outer membrane protein,OMP),위침대다충Pg설계구유교차보호작용적역묘제공후선파항원.방법 초고속리심법제취Pg301、PgATCC33277화PgW83균주OMP,응용PF2D목표단백쾌속분리계통분리OMP,대비분석득도공동OMP,용MALDI-TOF/TOF-MS천련질보결합수거고수색,학정단백질일급결구.결과 제일유색보취초분리,3주Pg공획득99개단백질양본;선정OMP조분B7,진행제이유반상색보분리,3주Pg공학정료8개공동적단백색보봉,운용MALDI-TOF/TOF-MS대단백양품진행감정,결과 학정료기중일개위이지적단백정안산-아간소A.결론 PF2D분리계통분변솔고、중현성호,결합MALDI-TOF/TOF-MS천련질보기술,가용우감정Pg적공동OMP.
Objective To screen a variety of Porphyromonas gingivalis (Pg) common outer membrane proteins with two-dimensional liquid phase fractionation (PF2D) and matrix-assisted laser desorption ionization time-of-flight/time-of-flight mass spectrometry ( MALDI-TOF/TOF-MS ) and provide candidate target antigen for the design of vaccines with cross protection against a variety of Pg. Methods The outer membrane proteins of Pg301, PgATCC33277 and PgW83 were extracted through ultracentrifugation, and then they were separated by ProteomeLab PF2D protein fractionation system. After separation, the outer membrane proteins were obtained through comparison, and the primary structure of the proteins was identified by MALDI-TOF/TOF-MS and database. Results Ninety-nine protein samples out of 3 strains of Pg were obtained after the high performance chromato focusing (HPCF) separation process. B7 fractions of 3 strains of Pg were separated by the reversed-phase high performance liquid chromatography (RP-HPCL) separation process. After comparison of peak and retention time of chromatogram, the 8 common protein peaks of 3 strains of Pg were confirmed. The protein samples were identified by MALDI-TOF/TOF-MS, and one of them was known protein arg-gingipainA. Conclusions PF2D protein fractionation system is of good reproducibility and high resolution. A combination of PF2D and MALDI-TOF/TOF-MS can be used to identify the common outer memberane proteins of Pg.