大肠杆菌K1致病株外膜蛋白T体外功能  

In vitro function of outer membrane protease T of Escherichia coli K1 pathogenic strain

作　　者：惠长野[1] 郭妍[2] 吴书驰[1] 彭亮[1] 曹虹[1] 黄胜和[1] 

机构地区：[1]南方医科大学公共卫生与热带医学学院微生物学系,广州510515 [2]中国科学院深圳先进技术研究院,深圳518054

出　　处：《微生物学报》2010年第1期48-53,共6页Acta Microbiologica Sinica

基　　金：国家自然科学基金(30972637)~~

摘　　要：纤溶酶原激活及水解抗菌肽利于致病菌侵袭及体内存活。【目的】构建大肠杆菌K1致病株E44的ompT基因缺失突变株,证实E44外膜蛋白T(OmpT)体外激活纤溶酶原及水解抗菌肽鱼精蛋白的活性。【方法】采用基因同源重组技术敲除大肠杆菌K1株E44中的ompT基因,构建ompT缺失突变株;二步柱层析纯化E44外膜组分,S-2251发色底物法测定其纤溶酶原激活活性;考察野生株E44、ompT基因敲除株E44ompT及转化带有ompT完整阅读框的质粒pUCT的E44ompT/pUCT三者对0.1mg/mL阳离子抗菌肽鱼精蛋白的敏感程度。【结果】利用自杀性载体pCVD442和同源重组的原理构建E44的ompT基因敲除株E44ompT;纯化得到约37kDa的E44外膜组分,S-2251发色底物法证实其具有纤溶酶原激活活性,纤溶酶原激活与膜组分的加入量呈一定量效关系;与野生株E44相比,ompT敲除株E44ompT对0.1mg/mL鱼精蛋白敏感,转化入带有ompT完整序列的质粒pUCT有一定的回补作用,E44ompT部分恢复抗鱼精蛋白能力。【结论】外膜蛋白T在致病株E44中有表达,并具有激活纤溶酶原及水解鱼精蛋白的活性。Plasminogen activation and antimicrobial peptide hydrolysis contribute to pathogens invasion and survival in vivo. [ Objective] To demonstrate the expression of outer membrane protease T in E. coli K1 pathogenic strain E44, its activity of plasminogen activator and protamine hydrolysis. [ Methods ] After Benzamidine Sepharose Fast Flow and SOURCE 30Q chromatography, we got E44 outer membrane mixed fraction, and examined its activity of plasminogen activation with chromogenic substrate S-2251 method. An ompT deletion mutant of E44 was constructed by using the suicide vector pCVD442, termed as E44ompT. We examined 0.1 mg/mL cationic antimicrobial peptide protamine susceptibility of E44, ompT mutant strain E44ompT and E44ompT harboring pUCT, which was constructed by inserting complete ompT open reading frame into pUC13. [ Results] We got about 37 kDa E44 membrane extract, which could activate plasminogen, and activation was membrane extract dose dependent. This confirmed the expression of outer membrane protease T in the outer membrane of E44. E44ompT was more susceptible to 0. 1 mg/mL protamine than E44, and E44ompT was partially complemented by pUCT. [ Conclusion] Outer membrane protease T is expressed in E. coli K1 pathogenic strain E44, and can activate plasminogen and hydrolyze protamine.

关 键 词：外膜蛋白T 纤溶酶原 激活 鱼精蛋白 水解 

分 类 号：Q93[生物学—微生物学]