大肠埃希菌蛋白指纹图谱的建立

Establishment of protein profiling of Escherichia coli

目的建立大肠埃希菌（Escherichia coli，E．coli）蛋白指纹图谱，为Ecoli感染快速诊断奠定基础。方法收集临床分离E.coli88株，提取细菌DNA，PCR检测Ecoli 16S rRNA。蛋白提取液提取细菌蛋白，干化学法测蛋白浓度，应用表面增强激光解析电离飞行时间质谱技术（SELDI-TOF-MS）检测Ecoli蛋白，采用Ciphergen Pro-teinchip软件自动采集数据。重复测定20次Ecoli混合标本，评价SELDI检测Ecoli蛋白分子量的重复性。结果E．coil标准菌株ATCC 25922和临床分离株均可检出16S rRNA。AU芯片能捕获近30个E．coli蛋白峰，其中19个蛋白峰构成E．coli特征性蛋白指纹图谱，各蛋白峰在临床分离E．coli间分子量变异系数≤0．2％。SELDI重复检测20次E．coli混合标本显示同一蛋白峰的分子量变异系数≤0．05％。结论E．coli在分子量3～20kD范围内具有特征性蛋白指纹图谱，为快速诊断E.coli感染提供了新思路。

Objective To establish protein profding of Escherichia coli（ E. coli） which lays a foundation for rapid diagnosis of E. coli infection. Methods Eighty-eight clinical isolates were collected. DNA and proteins of E. coli were extracted. The bacteria were identified as E. coli by 16S rRNA PCR,and protein concentrations were detected by dry chemistry method. The proteins of E. coli were analyzed by Surface enhanced laser desorption/ionization time-of-flight mass spectra （SELDI-TOF-MS）, and the data was collected by Ciphergen ProteinChip software. The mixed proteins of E. coli were measured for twenty times by SELDI to evaluate the reproducibihty. Results PCR results showed that 16S rRNA was found in standard strain ATCC 25922 and all clinical isolates of E. coli. Nearly thirty proteins were captured on AU chip, and the pro- tein profiling of E.coli was composed of nineteen proteins among them. The CV of protein biomarkers in molecular weight was less than 0.2% in clinical isolates, and it was less than 0.05 % in the mixed proteins. Conclusion There is a characteristic protein profiling in E. coli .which provides new approach for ranid diagnosis of E. coli infection.