铜绿假单胞菌Quorum-Sensing系统中rhlR基因的克隆表达及其免疫保护性研究  

The Cloning and Expression of rhlR, a Quorum Sensing Gene in Pseudomonas aeruginosa PAO1, and a Study of Its Effect on Bacterial Clearance Rate in Mouse Lung

作　　者：杜宝中[1] 谢轶[2] 贾文祥[1] 杨发龙[1] 曾蔚[1] 杨维青[1] 程曦[1] 陈恬[1] 张再蓉[1] 

机构地区：[1]四川大学华西基础医学与法医学院微生物学教研室,成都610041 [2]四川大学华西医院实验医学科

出　　处：《四川大学学报（医学版）》2006年第5期661-665,共5页Journal of Sichuan University(Medical Sciences)

基　　金：国家自然科学基金(批准号30370079);纽约中华医学会基金(CMBNo.82-412)资助

摘　　要：目的研究铜绿假单胞菌rhlR表达产物的分子生物学特性,以及对小鼠的免疫保护作用。方法以铜绿假单胞菌标准株PAO1的基因组DNA为模板,PCR方法扩增rhlR基因。利用pGEX4T-1载体构建rhlR-pGEX4T-1重组质粒,在大肠杆菌BL21(DE3)中诱导表达,并用免疫印迹验证。同时用纯化的重组蛋白免疫小鼠,菌落计数法检测免疫组和对照组鼠肺对铜绿假单胞菌的清除率。结果rhlR的全基因序列为726bp,经序列分析和同源性比较,与GenBank中的rhlR基因(登录号:AE004768)完全一致。大肠杆菌BL21(DE3)转化重组质粒rhlR-pGEX4T-1后,经IPTG诱导和SDS-PAGE分析,表达的融合蛋白质相对分子质量为54×103,其中RhlR蛋白为27×103。体内实验中,免疫小鼠肺部对铜绿假单胞菌的清除率与未经免疫的正常组比较,差异有统计学意义(P<0.05)。结论构建的rhlR-pGEX4T-1重组质粒能够在大肠杆菌BL21(DE3)中成功地表达出具有生物学活性的RhlR蛋白。重组蛋白对小鼠表现出一定的保护作用。Objective To inquire about the molecular characteristics of rhlR, a Quorum Sensing gene in Pseudomonas aeruginosa （P. aeruginosa） PAO1, and to explore the immunogenicity of RhlR protein in mouse. Methods The rhlR gene of PAO1 was amplified by PCR and cloned into pGEX4T-1 plasmid. The recombination was expressed in E. coli BL21 （DE3） and analyzed by SDS-PAGE and Western blotting. The fusion protein （GST- RhlR） was purified by GST purification Kit and the purified protein was used to immunize mice. P. aeruginosa PA0315 was injected into mouse lung to explore the immuno-protection of the protein. Results The 726 bp DNA fragment of rhlR was amplified from PAO1 general DNA. The restriction enzyme map showed that the inserted part of rhlR-pGEX4T-1 was successfully constructed and the gene was 100% homologous to rhlR in GenBank. The recombinant plasmid expressed a 54 kDa fusion protein （rhlR-GST） in E. coli BL21 （DE3） after induction by IPTG. The fusion protein could be recognized by mouse polyvalent antiserum against P. aeruginosa. The results showed that the bacterial clearance rate in mouse lung was 86. 92% in rhlR groups and 49. 44% in the control group. Conclusion A 54 kDa protein （RhlR-GST） has been successfully expressed in E. coli BL21 （DE3）. The RhlR could increase the bacterial clearance rate in mouse lung and may serve as immunoprotective antigen to develop the genetic engineering vaccine.

关 键 词：铜绿假单胞菌 基因表达 rhlR基因 免疫原性 

分 类 号：R392[医药卫生—免疫学] Q78[医药卫生—基础医学]