摘要
目的:克隆并鉴定6种常见呼吸道感染细菌16s rRNA基因,为制备基因芯片探针做准备。方法:设计、合成6种细菌16s rRNA基因扩增引物;PCR扩增16s rRNA基因目的片段;克隆16s rRNA基因片段;最后对重组质粒进行鉴定。结果:(1)6种细菌16s rRNA基因片段的PCR扩增结果:大肠埃希菌、金黄色葡萄球菌、肺炎链球菌、肺炎克雷伯杆菌、流感嗜血杆菌在1300bp处出现特异的目的片段;铜绿假单胞菌在1100bp出现特异的目的片段,与预计的片段大小吻合。(2)PCR产物分别与pMD18-T载体连接并转化JM109受体菌之后在Ampr培养基表面生长,其中蓝色菌落为阴性克隆,白色菌落是阳性克隆。(3)各克隆质粒PCR鉴定及双酶切鉴定,结果均可见特异目的带。(4)克隆质粒的序列分析示:6种细菌克隆质粒部分16s rRNA基因序列与GenBank中发表序列同源性为99.8%以上,说明细菌16s rRNA基因已克隆成功。结论:成功克隆了大肠埃希菌、金黄色葡萄球菌、肺炎链球菌、肺炎克雷伯杆菌、流感嗜血杆菌及铜绿假单胞菌16s rRNA基因片段,为制备基因芯片探针奠定了基础。
Objective：To clone and characterize the 16S rRNA of six species in the bacteria infecting respiratory tract to make gene chip. Methods：The primers of the target gene were designed and synthesized, and then the aimed fragment of the 16s rRNA was amplified by PCR and cloned. Finally the recombinant plasmids were characterized. Results： （ 1 ） The 16s rRNA gene of six species of bacteria was amplified. It was found that the size of amplified product by PCR was 1 300 bp in E. coli, S. aureus, S. pneumoniae, K. pneumoniae and H. influenzae and that of 1 100 bp in P. aeruginosa. （2）The JM109 transferred by the recombinant plasmid pMD18-T grew in Amp^r culture was white colonies. （3）The specific bands could be found by restriction endonuclease and PCR analysis. （4）The sequence of the six bacterial 16s rRNA showed the same as those in the GenBank. Conclusion：The 16s rRNA of six species of bacteria is successfully amplified and cloned into plasmid pMD18-T. It will provide the basis for making gene chip detecting the six species of bacteria infecting respiratory tract.
出处
《中国免疫学杂志》
CAS
CSCD
北大核心
2007年第7期641-643,647,共4页
Chinese Journal of Immunology
基金
吉林省科学技术厅资助项目(20010424)
