基于铜绿假单胞菌CRISPRs/Cas系统构建靶向oprM基因的siRNA表达载体及其抑制铜绿假单胞菌耐药性的探讨

Pseudomonas aeruginosa (PA) is one of most common drug-resistant bacteria in clinical.The increasing of drug resistance in PA is closely related to its active efflux system and has been a very thorny issue for clinical treatment of PA infection. Efflux pump inhibitors can effectively reduce PA drug resistance, but still easily induce PA to acquire drug resistance again, therefore, the new method or new way that can sustainedly inhibit PA drug resistance and is not easily induced PA to acquire drug resistance again urgently need to explore. Recent researches have found that most bacteria have CRISPRs/Cas defense system which is against intruders by similar RNAi mechanism. However, no research about utilizing the CRISPRs/Cas systems of bacteria to inhibit their drug resistance has been reported up to now. According to structural and functional characteristics of PA CRISPRs/Cas system,this project intends to imitate CRISPR unit to design repeat-siRNA-repeat fragment targeting PA active efflux system oprM gene which plays an important role in PA drug resistance and is then cloned into the plasmid vector, then PA is transformed by the constructed vector, and the change of PA drug resistance is observed to investigate whether the plasmid vector can inhibit PA drug resistance. If this study succeed, it will provide new approach for inhibition of bacterial resistance and investigation of bacterial gene function, and also will provide new idea for the development of new anti-bacterial drugs.

铜绿假单胞菌（PA）不断增强的耐药性已成为临床治疗PA感染极为棘手的问题。PA耐药与主动外排系统密切相关，外排泵蛋白抑制剂能有效降低PA耐药，但易诱导PA再次耐药，因此，迫切需要进一步探索能有效抑制PA耐药的新方法。研究发现大部分细菌都拥有通过类似RNAi机制抵御入侵者的CRISPRs/Cas防御系统，然而，至今未见利用细菌CRISPRs/Cas系统抑制其耐药的报道。本项目以oprM基因作为靶基因，模拟PA CRISPR单位的结构特点构建携带重复子-siRNA-oprM-重复子片段的重组质粒，转化PA，通过基因表达检测、药敏试验、动物实验等检测oprM基因的表达及PA耐药性的改变，探讨基于PA CRISPRs/Cas系统构建的靶向oprM基因的重组质粒抑制oprM基因表达及PA耐药性的作用和机制。本研究如获成功，将为细菌耐药性抑制、细菌基因功能研究、新型抗菌药物研发提供新思路和新方法。

铜绿假单胞菌（Pseudomonas aeruginosa,PA）耐药及治疗日益严峻，已成为目前临床的研究热点及难点。PA的主动外排系统是其主要的耐药机制之一，其oprM基因编码的OprM蛋白在PA的耐药中发挥着重要作用。研究发现oprM基因高表达株在多重耐药PA中的检出率明显高于非多重耐药菌株，提示oprM基因高表达与PA的多重耐药性相关。因此，降低PA的耐药性可以通过抑制或敲出oprM基因来恢复其敏感性。本研究通过研究siRNA干扰及构建铜绿假单胞菌CRISPR系统，将铜绿假单胞菌CRISPR单位的间隔子置换成在细菌耐药中发挥重要作用的基因片段，通过建立携带靶向oprM基因的重复子-siRNA-oprM-重复子片段的重组质粒，转化铜绿假单胞菌后，检测oprM基因表达及铜绿假单胞菌耐药性的变化，探讨重组质粒抑制oprM基因表达及证实siRNA质粒表达载体对oprM基因表达有无抑制作用；课题组已设计和筛选出有效的靶向oprM基因的siRNA片段，构建出表达的siRNA-质粒表达载体，初步阐明CRISPR系统敲出oprM基因以及siRNA靶向抑制铜绿假单胞菌oprM基因表达可以恢复铜绿假单胞菌的耐药性，为细菌耐药性抑制机制、新型抗菌药物的研发提供广阔的发展前景。