临床分离嗜麦芽窄食单胞菌喹诺酮耐药基因、产氨基糖苷类修饰酶耐药基因的研究免疫学专业论文.docxVIP

广州医 广州医学院硕士学位论文 嗜麦芽窄食单胞菌 3 个耐药基因的序列分析及原核表达 - - PAGE 10 - 结果： 以染色体DNA为模板，PCR扩增出三个大小分别约650bp、800bp和520bp左右 的片段并将其成功克隆入T载体。序列比对分析显示Smqnr开放阅读框长660bp， 编码24.3kDa蛋白；APH(3’’)-Ⅰ开放阅读框长813bp，编码29.9kDa蛋白； AAC(2’)-Ⅰ开放阅读框长549bp，编码20.2kDa蛋白。三个基因的序列已登录 GenBank，登录号分别为HQ315851、HQ315852和HQ315853。成功构建高效表达载 体pGEX-4T-1-Smqnr/ APH(3’’)-Ⅰ/AAC(2’)-Ⅰ，经IPTG诱导分别获得分子量 约50kDa、56kDa、46kDa的融合表达蛋白，与预期分子量相符。 结论： 成功构建了嗜麦芽窄食单胞菌喹诺酮耐药基因Smqnr、产氨基糖苷类修饰酶 基因APH(3’’)-Ⅰ和AAC(2’)-Ⅰ的T载体及高效原核表达载体pGEX-4T-1- Smqnr/APH(3’’)-Ⅰ/AAC(2’)-Ⅰ，IPTG诱导获得3个重组蛋白的大量表达,为了 解上述表达产物的特性和功能，以及其对细菌耐药机制的研究奠定基础。 关键词： 嗜麦芽窄食单胞菌；Smqnr 基因；产氨基糖苷类修饰酶基因；克隆；表达 Study on quinolone、AMEs resistance genes in clinical isolates of Stenotrophomonas maltophilia Applicant: Xiaoshan Guan Advisor: Zhenwen Zhou Abstract Objective: Stenotrophomonas maltophilia (Sm), an aerobic, non-fermentative, Gram-negative rod, resides in the nature generally, like various source of water、milk、 soil、animal and human body; It is also found in hospital environments such as dialysis apparatus、centr-A/VP tester、artificial respiration device and ventilation pipe. Sm plays an important role as an opportunistic nosocomial pathogen in recent 10 years on account of its detection rates in hospitalized patients are growing obviously and it can cause a broad spectrum of nosocomial infectious, e.g: pneumonia, meningitis, endocarditis, cellulitis, urinary tract and wound infection, bacteremia and sepsis. The majority of Sm clinical isolates are intrinsically resistant to many antimicrobials, which caused great difficulties for clinical treatment. Therefore, the resistance mechanism and the dissemination of resistance genes of Sm have attracted wide attention, research on the field is of great importance in recent years. To clone and express 3 drug resistance genes Smqnr 、 APH(3’’)-Ⅰand AAC(2’)-Ⅰ of Sm, provide the material for further study about crystal structure of Smqnr protein, lay foundation to understand the characteristics and function of expression products