幽门螺杆菌Lpp20真核表达重组载体的构建及鉴定

目的以幽门螺杆菌外膜脂蛋白Lpp20为目的基因,构建H.pylori Lpp20基因的真核表达重组载体pcD-NA3.1( )-Lpp20,为H.pylori核酸疫苗的研制奠定实验基础。方法用Pri mer5.0软件分析GenBank中H.pylori外膜脂蛋白Lpp20基因序列,设计合成相应特异性引物,引入BamHⅠ和XhoⅠ酶切位点,以H.pylori26695株基因组为模板,PCR扩增Lpp20目的基因片段,定向插入真核表达载体pcDNA3.1( )多克隆酶切位点中,构建重组体pcDNA3.1( )-Lpp20;通过酶切分析,PCR鉴定及测序鉴定,筛选阳性重组体。结果以H.pylori26695株基因组DNA为模板扩增出特异的Lpp20基因片段,大小约为528bp;双酶切及测序鉴定证明成功构建了H.pylori真核表达重组载体pcD-NA3.1( )-Lpp20。结论PCR扩增得到了大小约为528bp的H.pylori Lpp20目的基因片段;并成功构建了pcD-NA3.1( )-Lpp20真核表达重组载体。

Construction and Identification of Eukaryotic Expression Recombinant Vector of Helicobacter Pylori Lpp20 Gene

Objective The recombinant plasmid containing the outer membrane lipoprotein Lpp20 gene of Helicobacter pylori(H.pylori)was constructed to provide a foundation for future development of H.pylori DNA vaccine.Methods Primers were designed by Primer 5.0 software according to H.pylori Lpp20 gene sequence provided from GenBank.Polymerase chain reaction(PCR)was used to amplify the Lpp20 gene.The fragment was subcloned into appropriate site of pcDNA3.1( )eukaryotic expression vector by restriction enzyme digestion and linking reactions.The positive recombinant was identified by restriction endonuclease digestion,PCR amplification and sequencing.Results A 528bp Lpp20 specific gene fragment was amplified.Restriction enzyme analysis and sequencing showed that the eukaryotic expression recombinant pcDNA3.1( )-Lpp20 was successfully constructed.Conclusion The Lpp20 gene fragment(528bp)is successfully amplified,and the eukaryotic expression recombinant pcDNA3.1( )-Lpp20 is successfully constructed.