幽门螺杆菌ureI和lpp20乳酸乳球菌表达载体的构建及鉴定  

Construction and Identification of Lactococcus lactis Expression Vectors Carrying Helicobacter Pylori ureI and lpp20 Genes

作　　者：于文[1] 靖吉芳[1] 张艳[1] 

机构地区：[1]南华大学病原生物学研究所,湖南衡阳421001

出　　处：《实用预防医学》2009年第6期1702-1705,共4页Practical Preventive Medicine

基　　金：湖南省自然科学基金优秀面上项目(06JJ2093);湖南省教育厅青年基金(08B067)

摘　　要：目的以幽门螺杆菌尿素通道蛋白ureI和外膜脂蛋白lpp20为目的基因,分别构建乳酸乳球菌表达重组载体pNZ8112/ureI和pNZ8112/lpp20,为H.pylori乳酸乳球菌活菌口服疫苗的研制奠定实验基础。方法根据幽门螺杆菌尿素通道蛋白UreI和脂蛋白Lpp20的全基因序列,利用PRIMER5.0各设计一对引物,进行PCR扩增,获得ureI和lpp20目的基因片段,将其与分泌表达载体pNZ8112进行连接,通过电击转化进入宿主菌乳酸乳球菌NZ9000细胞内,通过酶切分析,PCR鉴定及测序鉴定,筛选阳性重组体,并进行重组疫苗菌稳定性检测。结果以H.pylori标准株基因组DNA为模板分别扩增出特异的ureI和lpp20基因片段,大小分别为588 bp和528 bp;双酶切及测序鉴定证明成功构建了H.pylori乳酸乳球菌表达重组载体pNZ8112/ureI和pNZ8112/lpp20。结论PCR扩增得到了大小约为588 bp和528 bp的H.pylori ureI和lpp20目的基因片段;并成功构建了幽门螺杆菌ureI及lpp20基因乳酸乳球菌表达重组载体。Objective To construct the recombinant plasmids containing the urel gene or Ipp20 gene of Helicobacter py- Iori （H. pylori）, and to lay a foundation for the further study of H. pylori live oral vaccine. Methods Primers were designed by PRIMER5.0 software according to the urel and Ipp20 genes sequences of H. pylori. The genes were amplified by polymerase chain reaction （PCR）. The PCR products were subcloned into the expression vector pNZ8112 to generate recombinant plasmids pNZ8112/urel and pNZ8112/Ipp20. Then the recombinant plasmids were transformed into Lactococcus lactis （L. lactis）NZ9000 by electroporation. The recombinant plasmids were identified by restriction endonuclease analysis, PCR and DNA sequencing. Segregational and structural stability of the recombinant plasmids in L. lactis were determined. Re- sults The Ipp20 and urel specific gene fragments were amplified. Restriction enzyme analysis and sequencing showed that the expression recombinant plasmids pNZ8112/urel and pNZ8112/Ipp20 were successfully constructed. Conclusions The expression recombinant plasmids pNZ8112/urel and pNZ8112/Ipp20 are successfully constructed. And there is no loss （100 % stability） of the recombinant plasmids in L. lactis NZ9000.

关 键 词：幽门螺杆菌 乳酸乳球菌 尿素通道蛋白UreⅠ 脂蛋白Lpp20 口服疫苗 

分 类 号：R379.9[医药卫生—病原生物学]