淋病奈瑟菌表面蛋白A真核表达载体的构建及表达

目的构建淋病奈瑟菌外膜蛋白———奈瑟菌表面蛋白A(neisseria surface protein A,NspA)的真核表达载体并使其在真核细胞中表达。方法用PCR方法扩增NspA基因,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。以脂质体法转染RAW264.7和COS-7细胞,用RT-PCR检测NspA mRNA的水平,免疫组化染色法检测蛋白表达。结果扩增的目的基因包括了全段NspA基因,长度约525bp。以脂质体转染RAW264.7和COS-7细胞后,用RT-PCR和免疫组化染色法检测表明,细胞可转录和表达NspA。结论成功构建了重组真核表达载体pcD-NA3.1(+)/NspA,并在真核细胞中得到表达,为研究此蛋白的免疫原性以及淋病基因疫苗的研制提供了物质基础。

Construction and Expression of the Eukaryotic Expression Vector Containing Gene of Neisseria Gonorrhoeae Surface Protein A

Objective To construct the eukaryotic expression vector which expressing the protein of NspA in mammalian cells.Methods The NspA gene was amplified by PCR.PCR product was cloned into sequencing vector pUCm-T and then subcloned into eukaryotic expression vector pcDNA3.1(+).Then the constructed plasmid was transfected into RAW264.7 and COS-7 cells by liposome-mediated gene transfer method.Level of NspA mRNA in transfected RAW264.7 cells was assayed by RT-PCR and NspA protein expressed in transfected COS-7 cells was detected by immunohistochemical staining.Results The amplified gene,which was about 525bp,included entire gene of NspA.The recombinant plasmid could express NspA protein with activity in mammalian cells.Conclusion The construction and expression of pcDNA3.1(+)/NspA have been achieved successfully,Which laid the foundation for studying immunogenicity of NspA and Neisseria gonorrhoeae DNA vaccine.