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

摘要:

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0.1 mg/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37 kDa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0.1 mg/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。

Abstract:

Abstract：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.