pcDNA3.1(+)/NspA真核表达载体的构建及鉴定  

Construction and Identification of Eukaryotic Expressing Vector of pcDNA3.1(+)/NspA

作　　者：谢良伊[1] 胡四海[1] 唐湘云[2] 杨胜辉[1] 余敏君[1] 

机构地区：[1]南华大学病原生物研究所,湖南衡阳421001 [2]湖南环境生物职业技术学院附属医院

出　　处：《实用预防医学》2006年第1期8-10,共3页Practical Preventive Medicine

基　　金：湖南省自然科学基金(05JJ30045)

摘　　要：目的构建pcDNA3.1(+)淋病奈瑟菌外膜蛋白—奈瑟菌表面蛋白A(Neisseria surface Protein A,NspA)基因真核表达载体,为研制淋病奈瑟菌核酸疫苗奠定基础。方法根据淋病奈瑟菌NspA基因序列,设计合成一对引物,用PCR方法从淋病奈瑟菌基因组DNA中扩增出NspA基因片段,将扩增的产物连接于测序载体pUCm-T上,并构建重组体pcDNA3.1(+)/NspA,进行酶切、PCR及测序鉴定。结果NspA基因体外扩增产物大小约为525 bp。所构建的pcDNA3.1(+)/NspA重组体经双酶切及PCR鉴定,与预期片断的大小相符;测序结果与GenBank中收录的NspA全长序列一致,表明pcDNA3.1(+)/NspA真核表达载体构建正确。结论成功地构建了淋病奈瑟菌真核重组表达载体pcD-NA3.1(+)/NspA,为NspA蛋白的功能研究和淋病奈瑟菌核酸疫苗的研制提供了物质基础。Objective To construct the recombinant eukaryotic expression vector pcDNA3. 1 （ ＋ ）/NspA, which provided the basic material for the development of Neisseria gonorrhoeae DNA vaccine. Methods A couple of primers were designed for PCR according to the known sequence of NspA. The NspA gene was amplified by PCR from genome of Neisseria gonorrhoeae WHO - A stain, and PCR product was cloned into sequencing vector pUOm - T and then subcloned into eukaryotic expression vector pcDNA3, 1 （ ＋ ）. The constructed pcDNA3, 1 （ ＋ ）/NspA was identified by restricting enzyme digestion analysis, PCR amplifying, and DNA sequencing. Results The size of amplified NspA gene was 525bp, Restriction enzyme digestion, PCR amplifying and DNA sequencing confirmed that pcDNA3, 1 （ ＋ ）/NspA had been constructed successfully, The harvested full- length sequence of NspA gene was identical with that registered in the GenBank, Conclusion The pcDNA3.1 （ ＋ ）/NspA has been successfully constructed, which provides the basic material for further study the function of NspA protein and the development of Neisseria gonorrhoeae DNA vaccine.

关 键 词：淋病奈瑟菌 表面蛋白A PCDNA3.1(+) 克隆 疫苗 

分 类 号：R378.16[医药卫生—病原生物学]