空肠弯曲菌Pen:19cadF基因真核表达重组质粒的构建与表达

Construction and Expression of the eukaryotic expression recombinant plasmid of CadF gene of Campylobacter jejuni Pen: 19

目的:构建空肠弯曲菌Pen:19 cadF基因真核表达重组质粒,并证实其能在COS-7细胞中表达。方法:以含有KpnI和EcoRI酶切位点的同一对引物行PCR反应,获得空肠弯曲菌Pen:2、Pen:8、Pen:19和Pen:21 cadF基因DNA片段。并选择与格林-巴利综合征最为相关的空肠弯曲菌Pen:19 PCR产物为目的基因片段,插入到真核表达载体pcDNA3.1(+)中,以构建重组质粒pcDNA3.1(+)-cadF。重组质粒经双酶切、PCR反应鉴定和DNA双向测序确认后转染至COS-7细胞。运用RT-PCR方法检测重组质粒在细胞m RNA水平的表达。结果:双酶切反应、PCR反应和DNA双向测序证实cadF基因插入成功。采用RT-PCR方法能够从重组质粒转染的COS-7细胞中扩增出与目的基因大小一致的DNA片段。结论:空肠弯曲菌Pen:19 cadF基因真核表达重组质粒pcDNA3.1(+)-cadF构建成功,并能在COS-7细胞中表达。为空肠弯曲菌DNA疫苗的研究奠定了良好的基础。

Objective： To construct the eukaryotic expression recombinant plasmid of cadF gene of Campylobacter jejuni Pen： 19 and to identify its express in COS-7 cells. Methods： Total DNA of Campylobacter jejuni Pen：2,Pen：8 ,Pen：19 and Pen： 21 were used as tem- plates respectively and cadF gene DNA with KpnI and EcoRI sites were amplified by PCR reactions using same primer. Because of its closest association with Guillain-Barr 6 syndrome,the PCR product of Pen： 19 was selected as target gene and inserted into the eukaryotic expression vector pcDNA3.1 （＋） and the recombinant plasmid pcDNA3.1 （＋）-cadF was constructed. The recombinant plasmid was identified by restriction enzyme digestion and PCR reaction and was confirmed by sequencing. The recombinant plasmid was then transfected into mammalian cell COS-7 for mRNA expression and the expression were investigated by RT-PCR reaction. Results： It was confirmed with restriction enzyme digestion and PCR reaction and sequencing that cadF gene was cloned into pcDNA3.1 （＋） successfully . The objective gene was amplified from COS-7 cell transfected with the recombinant plasmid pcDNA3.1 （ ＋ ）-cadF by RT-PCR. Conclusion： The eukaryotic expression recombinant plasmid pcDNA3.1 （ ＋ ）-cadF is constructed and expressed in COS-7 cell successfully. The results obtained lay the foundation for research on development of Campylobacter jejuni DNA vaccine.