摘要
目的针对空肠弯曲菌基因组序列为一超变量序列,测序比较空肠弯曲菌Pen∶2和Pen∶19peblA基因的同源性,以证实peblA基因的保守性。在此基础上,构建空肠弯曲菌Pen∶19peblA基因真核表达重组质粒。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∶21peblA基因DNA片段,测序比较Pen∶2和Pen∶19peblA基因的同源性。并选择与格林-巴利综合征最为相关的空肠弯曲菌Pen∶19peblA基因PCR产物为目的片段,插入到真核表达载体pcDNA3.1(+),以构建重组质粒。重组质粒经双酶切鉴定、PCR鉴定和测序确认后转染至COS-7细胞,RT-PCR方法检测其瞬时表达。对阳性克隆培养基上清中PEB1蛋白的表达用ELISA分析。结果测序证实空肠弯曲菌Pen∶19peblA基因序列与Pen∶2peblA基因序列一致。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。ELISA分析表明PEB1蛋白在COS-7细胞上清中获得表达。结论空肠弯曲菌peblA基因具有良好保守性,以此成功构建的重组质粒能在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 As the genome sequenee of Campylobacter jejuni is a hypervariable sequence, the peblA gene sequence of Campylobacterjejuni Pen: 19 was sequenced and the peblA gene conservative was identified. To construct the eukaryotic expression recombinant plasmid of the peblA gene of Campylobacter jejuni Pen: 19. Methods Total DNA of Campylobacter jejuni Pen:2, Pen: 8, Pen: 19 and Pen: 21 as templates respectively, peblA gene DNA with Kpn Ⅰ and EcoR Ⅰ sites was amplified by PCR using the same primer and the PCR product of Pen: 19 was sequenced. Because most close association with Guillain-Barre syndrome, the PCR product of Pen: 19 was selected as target gene and cloned into pcDNA3.1 ( + ) and constructed the recombinant plasmid that was identified by endonuclease digestion and PCR and confirmed by sequencing. The recombinant plasmid was then transfected into mammalian cell COS-7 for expression. The transient expression was investigated by RT-PCR. The expression of peblA gene in the culture supernatant of positive clones was analyzed by ELISA. Results The peblA gene sequence of Campylobacterjejuni Pen: 2 and Pen: 19 was identical. The target gene was amplified from COS-7 ceils transfected with recombinant plasmids by RT-PCR. PEBl protein could express in the culture supernatant in the transfected COS-7 ceils by ELISA. Conclusion The peblA gene of Campylobacterjejuni was conservative. The recombinant plasmid was constructed and expressed in COS-7 ceils successfully. The results obtained lay the foundation for research on development of Campylobacter jejuni DNA vaccine.
出处
《第三军医大学学报》
CAS
CSCD
北大核心
2006年第7期644-647,共4页
Journal of Third Military Medical University
基金
国家自然科学基金资助项目(30371500)~~