摘要
目的:构建不可分型流感嗜血杆菌(nontypeableHaemophilus influenzae,NTHi)外膜蛋白P6(outermembrane protein P6,P6)真核表达的重组质粒,并在HeLa细胞中表达,为核酸疫苗的开发奠定基础。方法:以NTHi基因组为模板,扩增编码P6蛋白的基因片段,酶切、纯化P6基因与真核载体pcDNA3.1/HisA后进行连接,之后转化并筛选含有目的基因的重组质粒pcDNA3.1/HisA-P6;将重组子转染至HeLa细胞,荧光蛋白法检测其表达产物。结果:经质粒PCR、酶切、测序证实插入的基因片段为NTHi-P6蛋白编码基因;荧光显微镜下显示,该重组质粒能够在HeLa细胞中表达目的蛋白。结论:成功地构建了真核重组质粒pcD-NA3.1/HisA-P6,并在HeLa细胞中表达。
Objective： The recombinant eukaryotic plasmids for gene encoding outer membrane protein P6（P6） of the nontypeable Haemophilus influenzae（NTHi） was constructed and expressed in HeLa cells for the sake of providing a foundation to develop the nucleic acid vaccine.Methods： The target gene fragment encoding the P6 was amplified from the genome of the NTHi which was used as the template,then the plasmids pcDNA3.1/HisA and the P6 fragment was digested with endonucleases simultaneously,purified and connected.Then the recombinant plasmids pcDNA3.1/HisA-P6 was used to select,transform and express in HeLa cells;meanwhile,the expression of this eukaryotic plasmids in HeLa cells was investigated by using fluorescence microscopy.Results： As demonstrated by PCR,restriction endonuclease analysis and sequencing,the size of the inserted target gene fragment was found to be P6 which was also proved to encode the P6 protein in HeLa cells by means of fluorescence microscopy.Conclusion： The recombinant eukaryotic plasmids pcDNA3.1/HisA-P6 was successfully constructed and expressed in HeLa cells.
出处
《东南大学学报（医学版）》
CAS
2011年第3期491-494,共4页
Journal of Southeast University(Medical Science Edition)
基金
河北省教育厅重点课题(Z2010116)
关键词
不可分型流感嗜血杆菌
外膜蛋白P6
核酸疫苗
真核表达质粒
nontypeable Haemophilus influenzae
outer membrane protein P6
nucleinic acid vaccine
eukaryotic expression plasmids
