摘要
目的构建尿路致病性大肠埃希菌Ⅰ型菌毛的fimH基因真核表达载体,为尿路致病性大肠埃希菌的核酸疫苗研制奠定基础。方法通过PCR扩增尿路致病性大肠埃希菌临床分离株的fimH全基因序列,克隆至pMD19-T载体,PCR、酶切及测序鉴定后,将fimH基因片段克隆至真核表达载体pcDNA3.0,构建pcDNA3.0-fimH重组质粒,并进行PCR和酶切鉴定。结果 PCR扩增尿路致病性大肠埃希菌fimH基因片段为910 bp;构建的pcDNA3.0-fimH重组质粒经BamHⅠ和XhoⅠ双酶切,产生1个与fimH基因PCR产物大小一致的小片段和1个不同于pcDNA3.0-fimH重组质粒的大片段,表明fimH基因已成功插入pcDNA3.0质粒中。结论成功构建尿路致病性大肠埃希菌fimH基因真核表达载体pcDNA3.0-fimH。
Objective To lay the foundation for the development of a uropathogenic Escherichia coli gene vaccine by constructing and identifying the eukaryotic expression vector pcDNA3.0-fimH for expression of the fimH gene from uropathogenic E.coli.Methods Gene fragments of fimH from uropathogenic E.coli 6704 were amplified by PCR.The purified PCR fragments were ligated into the pMD19-T simple vector,the positive clones were directly screened from bacteria and identified by PCR,and the PCR products were digested via double enzymes following sequencing of the recombinant plasmid.The fimH gene fragments from pMD19-T-fimH clones were cloned into the eukaryotic expression vector pcDNA3.0 using restriction enzymes.The recombinant plasmid pcDNA3.0-fimH was identified through PCR,enzyme digestion,and sequencing.Results An fimH gene fragment of about 910 bp was amplified.The recombinant plasmid pcDNA3.0-fimH was digested via double enzymes BamHⅠ and XhoⅠ to produce small fragments with the same size as PCR products of fimH and large varying fragments of pcDNA3.0-fimH.This indicated that the fimH gene was successfully inserted into the pcDNA3.0 plasmid.Conclusion The eukaryotic expression vector pcDNA3.0-fimH was successfully constructed for expression of fimH from uropathogenic E.coli.
出处
《中国病原生物学杂志》
CSCD
2011年第6期408-410,共3页
Journal of Pathogen Biology
