空肠弯曲菌Pen:19 peblA基因序列分析及其真核表达重组质粒的构建  

Analysis of peblA gene sequence and construction of eukaryotic expression recombinant plasmid of peblA gene of Campylobacter jejuni Pen:19

作　　者：郑惠[1] 蔡方成[1] 邓兵[1] 钟敏[1] 

机构地区：[1]重庆医科大学儿童医院神经内科,重庆400014

出　　处：《第三军医大学学报》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)~~

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.

关 键 词：空肠弯曲菌 peblA基因 重组质粒 DNA疫苗 

分 类 号：Q755[生物学—分子生物学] Q782