【摘要】 目的研究细胞间信息传递机制之一假单胞菌喹诺酮信号(PQS)在铜绿假单胞菌发病机制中的作用。方法基于基因融合分析在转录研究中的实用性,构建了两种PQS相关对照质粒和整合突变株。一种是酶切质粒pYHP441获得pqsA’-lacZ片段后,亚克隆入质粒m in iCTX-1中,构建成pqsA’的阳性表达质粒,随后将构建的质粒,通过双亲交配过程整合入野生型铜绿假单胞菌株PAO-1染色体组中;另一种是通过点特异插入诱变策略,将四环素基因盒插入启动子pqsD和pqsE之间,构建的阴性质粒转化入大肠杆菌S17-1株后,和上述pqsA’阳性表达突变株进行双亲交配过程。结果针对今后PQS的相关研究,构建得到稳定表达PQS的质粒和阳性突变株作为阳性对照。构建获得pqsA-E启动子敲除质粒和相应PQS表达受阻的整合突变株作为阴性对照。结论两种质粒和整合突变株的构建成功,使得PQS的相关研究结论具有客观性、精确性和有效性。更多还原

【Abstract】 Objective To study the unique cell-to-cell communication mechanism of Pseudomonas quinolone signal(PQS) in the pathogenesis of Pseudomonas aeruginosa. Methods Based on the usefulness of gene fusion analysis for transcriptional analysis,two kinds of plasmids and integrant mutant strains were developed for the systemic study of PQS.One was the pqsA’ positive expression plasmid constructed by cloning the pqsA’-lacZ fusion digested from pYHP441 into miniCTX-1,whose sequence was then integrated into wild type PAO-1 chromosome by biparental mating procedure.The other was pqsA-E operon knock-out plasmid whose sequence between pqsD and pqsE operon was inserted by tetracycline cassette by the site specific insertional mutagenesis strategy and the mutant constructed by biparental mating of S17-1 that harbored the plasmid and pqsA’ positive expression mutant. Results(We got) the positive palsmid and integrant mutant strain which expressed PQS stably and could be used as a positive control,and the pqsA-D knock-out plasmid and integrant mutant strains with disturbed PQS expression which could be used as a negative control for β-galactosidase analysis. Conclusion The successful development of the palsmids and integrant mutant strains can make the analysis of PQS study objectively,accurately and efficaciously.更多还原