嗜麦芽窄食单胞菌产氨基糖苷类修饰酶基因APH(3′′)-Ⅰ和AAC(2′)-Ⅰ的克隆与表达  

Clone and expression of APH(3′′)-Ⅰand AAC(2′)-Ⅰ gene of Stenotrophomonas maltophilia

作　　者：关小珊 关锐梨 钟华敏 邓秋连 谢永强 周珍文 

机构地区：[1]广州市妇女儿童医疗中心儿童院区检验科,510120

出　　处：《国际检验医学杂志》2016年第15期2099-2101,共3页International Journal of Laboratory Medicine

摘　　要：目的对嗜麦芽窄食单胞菌临床分离株gzch810(SM gzch810)携带的产氨基糖苷类修饰酶基因APH(3′′)-Ⅰ和AAC(2′)-Ⅰ进行扩增、测序及原核表达,为下一步功能试验的开展提供基础材料。方法提取SM gzch810基因组染色体,PCR扩增APH(3′′)-Ⅰ和AAC(2′)-Ⅰ全基因并克隆入pMD18-T载体进行核苷酸序列分析;将APH(3′′)-Ⅰ和AAC(2′)-Ⅰ基因克隆至表达载体pGEX-4T-1,转化大肠杆菌BL21,SDS-PAGE分析融合蛋白表达情况。结果以染色体DNA为模板,成功扩增800bp APH(3′′)-Ⅰ基因和550bp AAC(2′)-Ⅰ基因;序列比对分析显示其与相关报道序列核苷酸和氨基酸一致性分别达91%及95%;SM gzch810APH(3′′)-Ⅰ和AAC(2′)-Ⅰ序列已登录GenBank(登录号:HQ315852和HQ315853);SDS-PAGE显示,融合基因表达的蛋白相对分子质量分别约为56×103和46×103。结论从SM gzch810中成功克隆及表达了APH(3′′)-Ⅰ和AAC(2′)-Ⅰ基因,为下一步检测上述两种重组体大肠杆菌对相应抗菌药物的耐药性及其功能评价做好准备。Objective To perform the amplification,sequencing and prokaryotic expression of APH（3′′）-Ⅰand AAC（2′）-Ⅰgenes from the clinically isolated gzch810strain（SM gzch810）of Stenotrophomonas maltophilia to provide the basic materials for the next step functional test.Methods The SM gzch810 genome chromosome was extracted,the APH（3′′）-Ⅰ,AAC（2′）-Ⅰ whole genes were amplified by PCR and sequenced after being cloned into pMD18-T vector.The recombination were subcloned into pGEX-4T-1vector and the expression of the recombinant APH（3′′）-Ⅰand AAC（2′）-Ⅰ were analyzed by SDS-PAGE.Results The800 bp and 550 bp DNA fragments of APH（3′′）-Ⅰ,AAC（2′）-Ⅰ gene were amplified from SM gzch810 by PCR and sequenced;the sequence comparison analysis showed that DNA and amino acid sequence identities of APH（3′′）-Ⅰand AAC（2′）-Ⅰ genes with other strains were 91% and 95% respectively.The sequence of APH（3′′）-Ⅰand AAC（2′）-Ⅰ of SM gzch810 were submitted to GenBank（accession number：HQ315852and HQ315853）;two major protein bands corresponding to the expected recombinant GSTTP fusion proteins（56×103 and 46×103 respectively）were identified by SDS-PAGE.Conclusion APH（3′′）-Ⅰand AAC（2′）-Ⅰgene of SM gzch810 are successfully cloned and expressed,which lays a good foundation for further detecting corresponding antibiotic resistance and functional evaluation of above two kinds of recombinant E.coli.

关 键 词：嗜麦芽窄食单胞菌 产氨基糖苷类修饰酶基因 克隆 原核表达 

分 类 号：R440[医药卫生—诊断学]