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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 construct the eukaryotic expression vector of fimH gene of urinary tract pathogenic Escherichia coli type Ⅰ pili to lay the foundation for the development of DNA vaccine of pathogenic Escherichia coli in urinary tract. Methods The fimH gene of clinical isolates of pathogenic Escherichia coli was amplified by PCR and cloned into pMD19-T vector. After PCR, restriction enzyme digestion and sequencing, the fimH gene fragment was cloned into the eukaryotic expression vector pcDNA3 .0, construct pcDNA3.0-fimH recombinant plasmid, and PCR and restriction enzyme digestion. Results The fimH gene fragment of pathogenic Escherichia coli was amplified by PCR. The fragment was 910 bp. The recombinant plasmid pcDNA3.0-fimH was digested with BamHⅠ and XhoⅠ to generate a small fragment with the same size as the fimH gene PCR product And a large fragment different from the pcDNA3.0-fimH recombinant plasmid, indicating that the fimH gene has been successfully inserted into the pcDNA3.0 plasmid. Conclusion The eukaryotic expression vector pcDNA3.0-fimH of fimH gene of urinary pathogenic Escherichia coli was successfully constructed.