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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apr A、apr I和flic基因,构建重组表达载体p ET-28a-AprA、p ET-28a-AprI和p ET-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。
OBJECTIVE: To construct E. coli BL21 (DE3) recombinant adenovirus expressing AprA, AprI and flagellin, which are important virulence factors of Pseudomonas aeruginosa (PAE) And the biological activity of AprI. Methods The recombinant plasmids p ET-28a-AprA, p ET-28a-AprI and p ET-28a-flagellin were amplified by polymerase chain reaction (PCR) Escherichia coli BL21 (DE3) was induced by IPTG, and the inclusion body was denatured and renatured to obtain AprA protein. Purified by nickel affinity resin, AprI and flagellin were obtained. AprA cleaves flagellin activity by SDS-PAGE. Results The recombinant protein AprA was expressed as inclusion bodies and AprI and flagellin were expressed as soluble forms. AprA cleaves flagellin and AprI inhibits AprA cleavage of flagellin. Conclusion AprA secreted by PAE has the activity of cleaving flagellin, which can be inhibited by AprI. This study lays a foundation for further study on the pathogenesis of AprA in PAE.