Immunoproteomics of membrane proteins of Shigella flexneri 2a 2457T

AIM: To screen the immunogenic membrane proteins of Shigella flexneri 2a 2457T. METHODS: The routine two-dimensional polyacrylamide gel electrophoresis (2-DE) and Western blotting were combined to screen immunogenic proteins of S. flexneri 2a 2457T. Serum was gained from rabbits immunized with the same bacteria. Immunogenic spots were cut out from the polyacrylamide gel and digested by trypsin in-gel. Matrix-assisted laser desorption/ionization time of flight-mass spectrometry (MALDI-TOF-MS) was performed to determine the molecular weight of peptides. Electrospray ionization (ESI-MS/MS) was performed to determine the sequences of the interesting peptides. RESULTS: A total of 20 spots were successfully identified from Coomassie brilliant blue stained gels representing 13 protein entries, 5 known antigens and 8 novel antigens. A hypothetical protein (YaeT) was detected, which might be a candidate target of vaccine. CONCLUSION: Membrane proteins of 5. flexneri 2a 2457T were successfully observed by 2-DE. Several known and novel antigens were identified by mass spectrum.     

志贺菌属肠毒素ShET1/ShET2的基因型PCR分析

目的 对福氏2a志贺菌肠毒素ShET1和肠毒素ShET2进行基因分型，提高福氏2a志贺菌同源克隆鉴定系统的分析水平。方法 对93株分离自不同地区、不同时间的福氏2a志贺菌用PCR法检测志贺菌肠毒素ShET1／ShET2基因并进行基因分型。结果 93株福氏2a志贺菌按志贺菌肠毒素ShET1／ShET2基因分为4种基因型，即12株ShET1(-)／ShET2( )，14株ShET1( )／ShET2(-)，59株ShET1( )／ShET2( )，8株ShET1(-)／ShET2(-)。结论 福氏2a志贺菌肠毒素ShET1／ShET2基因PCR检测可用于福氏2a志贺菌的初步筛检。     

弗氏志贺菌ΔlysA突变株的构建及SILAC技术的应用尝试

目的构建弗氏2a志贺菌301株赖氨酸营养缺陷型突变株,为将细胞培养条件下稳定同位素标记（SILAC）技术应用于志贺菌蛋白质组学研究奠定基础。方法采用λ-Red重组系统对弗氏2a志贺菌301株lysA基因进行缺失,对野生株和突变株进行基本的表型测评和毒力评价,并利用双向电泳技术比较二者在全菌蛋白表达谱上的差异,最后将突变株分别在含有12C6-赖氨酸和13C6-赖氨酸的培养基中培养,根据双向电泳胶图取对应蛋白点进行胶内酶切及MALDI-TOF/TOF分析。结果成功构建了301 lysA基因缺失突变株,蛋白鉴定结果显示所取对应蛋白点为同一蛋白,在质谱图上轻重同位素峰位移为6 amu。结论本研究所构建的赖氨酸营养缺陷型突变株适合进行SILAC分析。     

上海市闵行区2007-2009年志贺菌菌群分布及其药敏分析

[目的]了解近年上海市闵行区志贺菌菌群分布和耐药性特点,为细菌性痢疾的临床合理用药和疾病预防控制工作提供科学依据。[方法]对2007-2009年该区各肠道门诊监测点腹泻病人的肛拭标本进行细菌分离培养、鉴定和血清分型,并用改良纸片扩散法法进行药物敏感试验。[结果]251株志贺菌中,B群福氏志贺菌180株、D群宋内志贺菌71株,未分离出其他群型的志贺菌;251株志贺菌对四环素、复方新诺明、氨苄西林、利福平和萘啶酸耐药明显,均〉76．00％,对环丙沙星、阿莫西林相对比较敏感,但B群与D群之间差异具有统计学意义;3年间,庆大霉素等药物的耐药性有所递增。[结论]该区志贺菌菌群具有较高的耐药性,且部分药物的耐药性有逐年递增的趋势,应加强耐药性监测和疾病防治。     

福氏志贺菌血清型单重PCR鉴定方法的建立及应用

目的 建立基于O抗修饰基因的福氏志贺菌血清型PCR鉴定方法.方法 根据福氏志贺菌O抗原合成及修饰特异基因设计引物,以常见的14种福氏志贺菌血清型(包括1a、1b、1c、2a、2b、3a、3b、4a、4b、5a、Y、X、Xv和F6)为标准菌株,建立福氏志贺菌血清型单重PCR鉴定方法;并以痢疾志贺菌、宋内志贺菌、鲍氏志贺菌和腹泻相关的其他菌属菌株验证特异性;应用该方法对106株福氏志贺菌临床分离株进行PCR血清分型.结果 建立一种福氏志贺菌血清型单重PCR鉴定方法,通过8个PCR反应,能够鉴定目前已知的15种血清型中的14种(Xv除外).检测灵敏度在10 pg至1 ng DNA(20μl反应体系).对106株福氏志贺菌临床分离株的检测结果提示,PCR分型方法与玻片凝集法具有很高的一致性.结论 本研究建立的福氏志贺菌血清型单重PCR鉴定方法具有特异性、灵敏性,可用于临床检测.