铜绿假单胞菌碱性蛋白酶AprA及其抑制剂AprI的表达、纯化和初步的活性验证

目的：利用大肠杆菌BL21（DE3）重组表达铜绿假单胞菌（Pseudomonas aeruginosa，PAE）重要毒力因子碱性蛋白酶（AprA）、碱性蛋白酶抑制剂（AprI）和鞭毛蛋白（flagellin），并检测AprA和AprI的生物学活性。方法以该菌PAO1的基因组为模板，PCR扩增aprA、aprI和flic基因，构建重组表达载体pET-28a-AprA、pET-28a-AprI和pET-28a-鞭毛蛋白，转化至大肠杆菌BL21（DE3），利用IPTG诱导表达，将包涵体变性、复性后纯化获得AprA蛋白，通过镍亲和树脂纯化获得AprI和鞭毛蛋白。利用SDS-PAGE检测AprA裂解鞭毛蛋白活性。结果重组蛋白AprA以包涵体的形式表达，AprI和鞭毛蛋白以可溶性形式表达。 AprA能裂解鞭毛蛋白，加入AprI可抑制AprA裂解鞭毛蛋白。结论 PAE分泌的AprA蛋白具有裂解鞭毛蛋白的活性，该活性能被AprI抑制。该研究为深入研究PAE的AprA的致病机制奠定了基础。

Expression,purification and detection of activities of AprA and its inhibitor AprI from Pseudomonas aeruginosa

Objective To clone and express the alkaline protease AprA , one important virulence factor secreted by Pseudomonas aeruginosa(PAE)in Escherichia coli, to clone and express the inhibitor of AprA (AprI) and its substrate flagellin , and to detect the function of AprA and the inhibitory function of AprI .Methods The genes encoding AprA ,AprI and flagellin gene were amplified respectively by PCR using PAE PAO 1 genome DNA as the template .The expression vec-tors (pET-28a-AprA, pET-28a-AprI and pET-28a-Flagellin) were constructed and transformed into E.coli BL21(DE3) respectively.The recombinant AprA protein was expressed by IPTG induction and purified via denaturing and renaturation. The recombinant AprI and flagellin were expressed and purified by Ni 2+affinity chromatography .The cleavage activities of AprA on flagellin were detected by SDS-PAGE.Results Recombinant AprA , AprI and flagellin protein were expressed and purified .It was demonstrated that AprA cleaved flagellin , which was blocked by AprI .Conclusion Recombinant AprA could cleave its substrates as an alkaline protease , and its inhibitor AprI inhibits the activities of AprA .This study will contribute to further investigations on the role of AprA in the pathogenesis of PAE .