pcDNA3．1（＋）／NspA真核表达载体的构建及鉴定

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。

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

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 pUCm-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.