鼠疫耶尔森氏菌Pla蛋白的分离纯化与质谱分析  

Purification and Mass spectrometry of the Plasminogen activator of Yersinia pestis

作　　者：杜春红[1] 王鹏[1] 张建中[2] 肖迪[2] 石丽媛[1] 唐雪[1] 宋志忠[1] 

机构地区：[1]云南省地方病防治所,云南大理671000 [2]中国疾病预防控制中心传染病预防控制所

出　　处：《中国地方病防治》2011年第3期161-165,共5页Chinese Journal of Control of Endemic Diseases

基　　金：云南省科技攻关项目(2007CA010)

摘　　要：目的建立一种从鼠疫耶尔森氏菌疫苗株EV76中分离纯化纤维蛋白酶原激活因子（Pla）的方法。方法采用超声破碎与硫酸铵盐析结合的方法初步提取Pla,经CHT陶瓷羟基磷灰石层析分离纯化,所获取的Pla进行质谱分析及Western blot实验。结果菌悬液超声破碎后上清以0~10%饱和硫酸铵沉淀,获得含有相对分子质量（Mr）约为31 000、35 000、37 000的3条Pla蛋白带,约占蛋白总量33%;CHT柱层析后,3条带约占蛋白总量80%。经质谱分析,在Mr约为31 000、35 000的条带中含有Pla,Pla单克隆抗体能特异性地识别该蛋白。结论采用超声破碎结合硫酸铵盐析的方法可从鼠疫菌中提取Pla,CHT柱层析可达到基本纯化,获得的样品经质谱分析和Western blot实验证实为Pla蛋白。Objective To develop a method to get purified plasminogen activator（Pla） from vaccine strain EV76 of Yersinia pestis.Methods Crude Pla was collected by sonication and precipitation with ammonium sulfate.Purified Pla was obtained by ceramic hydroxyapatite（CHT） chromatography,and it was testified by mass spectrometry analysis and Western blot.Results Three proteins with relative molecular mass（Mr） of approximately 31 000,35 000 and 37 000,which account total proteins for 33%,were obtained from bacterial suspension supernatant after sonication and precipitation with 0 to 10% saturated ammonium sulfate.Above three proteins account for 80% after CHT column chromatography.By mass spectrometry,proteins with the molecular mass of about 31 000,35 000 were identified as Pla,and the monoclonal antibody of Pla can specifically recognize the two proteins by WB.Conclusions Crude Pla can be extracted from Y.Pestis by sonication and precipitation with ammonium sulfate,and purified Pla can obtained by CHT column chromatography.The Pla protein was confirmed by mass spectrometry and Western blot.

关 键 词：鼠疫耶尔森氏菌 纤溶酶原激活因子 提取 纯化 质谱 

分 类 号：R516.8[医药卫生—内科学]