摘要
目的构建以尿路致病性大肠杆菌临床分离株Ⅰ型菌毛编码基因fimH为目的基因的真核表达载体pcDNA3.0-fimH。方法提取尿路致病性大肠杆菌临床株基因组DNA,PCR扩增fimH基因片段,然后克隆至TA载体,通过PCR、酶切及测序鉴定后,将fimH基因片段用限制性内切酶切下克隆至真核表达载体pcDNA3.0,构建pcDNA3.0-fimH重组质粒。通过PCR、酶切对重组质粒pcDNA3.0-fimH进行鉴定。结果 PCR法克隆出全长为903 bp的fimH基因并构建了pcDNA3.0-fimH重组质粒。结论本研究成功构建尿路致病性大肠杆菌Ⅰ型菌毛fimH基因真核表达质粒pcDNA3.0-fimH,为尿路致病性大肠杆菌基因疫苗的研制奠定了基础。
Objective To construct eukaryotic expression plasmid of fim H gene of clinical uropathogenic Escherichia coli typeⅠpili.Methods Genomic DNA of uropathogenic Escherichia coli was extracted and fimH gene fragment was amplified from genomic DNA of uropathogenic Escherichia coli by PCR.The purified PCR fragment was ligated into the pMD19-T simple Vector,and then the positive clones were screened and identified by PCR from bacteria directly and digested via double enzymes following with a sequencing of recombinant plasmid.fimH gene fragments from TA clones were cloned to eukaryotic expression vector pcDNA3.0 under the restriction enzyme.The recombinant plasmid pcDNA3.0-fimH was identified through PCR,enzyme digestion and sequencing.Results The fimH gene fragment was amplified correctly as the size of gene was about 903 bp and eukaryotic expression vector pcDNA3.0-fimH was constructed.Conclusion Type Ⅰ pili of pathogenic E.coli fimH gene eukaryotic expression vector pcDNA3.0-fimH was successfully constructed,the study laid the foundation for urinary tract pathogenic E.coli genes vaccine.
出处
《滨州医学院学报》
2010年第6期424-426,共3页
Journal of Binzhou Medical University
基金
滨州医学院科技计划项目(BY2007KJ42)
