铜绿假单胞菌外膜蛋白D_2与亚胺培南耐药的关系  

The relationship between outer membrane protein D_2 of Pseudomonas aeruginosa and imipenem resistance

作　　者：郭春霞[1] 贺永文[1] 潘延凤[1] 李淑莉[1] 王华[1] 

机构地区：[1]华中科技大学同济医学院附属协和医院传染科,武汉430022

出　　处：《中国感染与化疗杂志》2007年第5期335-338,共4页Chinese Journal of Infection and Chemotherapy

摘　　要：目的制备铜绿假单胞菌(PA)的外膜蛋白(Opr)D_2多克隆抗体,探讨OprD_2缺失与亚胺培南耐药的关系。方法常规酚-氯仿法提取PA的基因组DNA,PCR扩增OprD_2基因,构建原核表达质粒pRSET-OprD_2,在大肠埃希菌BL21(DE3)中表达目的蛋白,胶回收后制备兔多克隆抗体,应用该抗体检测临床分离的32株PA OprD_2的表达情况。结果成功构建了OprD_2原核表达载体,制备了特异性好的OprD_2多克隆抗体。蛋白印迹法检测27株临床分离的耐亚胺培南PA,20株(74.1%)OprD_2表达缺失,5株(18.5%)OprD_2表达明显减少,2株(7.4%)OprD_2表达正常。结论临床分离的PA OprD_2表达的缺失或减少是PA对亚胺培南耐药的主要机制之一。Objective To prepare specific polyclonal antibodies to outer membrane protein （Opr） D2 of Pseudomonas aeruginosa （PA）, and explore the relationship between loss of OprD2 and imipenem resistance. Methods The genomic DNA of PA was extracted with phenol： chloroform. OprD2 coding gene was amplified by PCR and prokaryotic expression vector pRSET-OprD2 was constructed. OprD2 protein was expressed by IPTG induction in E. coli BL21 （DE3）, and purified with SDS-PAGE. The new protein band was recovered and used as antigens to subcutaneously immunize two New Zealand rabbits to prepare polyclonal antibody. The specificity of the antibody was determined by Western blot. The expression of OprD2 in 32 clinical isolates of PA was detected with the prepared polyclonal antibody by Western blot. Results The vector pRSET-Opr1）2 has been successfully expressed in E. coli BL21 （DE3）. The polyclonal anti-OptD2 antibody with high specificity has been successfully prepared. Present results show that of the 27 imipenem-resistant PA clinical isolates, OprD2 protein was low-expressed in 5 isolates （18.5%） and normally expressed in 2 isolates （7. 4%） but not expressed in 20 isolates （74. 1%）. Conclusions The loss or low-expression of OprD2 is one of the essential mechanisms accounting for imipenem resistance in clinical isolates of PA.

关 键 词：铜绿假单胞菌 外膜蛋白 多克隆抗体 亚胺培南 耐药性 

分 类 号：R378[医药卫生—病原生物学]