牙龈卟啉菌菌毛FimA基因高效植物表达载体的构建  

Construction of a plant effective expression vector containing porphyromonas gingivalis fimbriae A gene

作　　者：戴海燕[1] 王华[2] 文少敏[1] 解娜[1] 邱瑾[1] 

机构地区：[1]海南医学院口腔医学院,海南海口570102 [2]海南医学院省热带病重点实验室,海南海口570102

出　　处：《牙体牙髓牙周病学杂志》2010年第11期630-633,共4页Chinese Journal of Conservative Dentistry

基　　金：海南省自然科学基金资助项目(807096)

摘　　要：目的:构建果实特异启动子驱动的含牙龈卟啉单胞菌菌毛FimA基因的高效植物表达载体,并进一步提高抗原基因的表达量和免疫原性,为研制有效的转基因植物牙周炎疫苗打下基础。方法:以提取的番茄基因组DNA为模板扩增番茄果实特异表达启动子E8的核心序列(约1.11kb),同时合成通过接头连接的霍乱毒素B亚基和FimA(266～337)(约600bp),并通过SOE PCR,即重叠区扩增基因拼接法(genesplicing by overlap extension)将两者拼接,拼接后的序列为EF。用EcoR I和Pst I分别双酶切EF序列及表达载体pCAMBIA1302,连接转化,得到的重组质粒pCAMBIA1302-EF用电击法转化根癌农杆菌GV3103。结果:重组质粒经PCR和酶切鉴定证明已成功转化。结论:本研究成功构建了番茄果实特异性启动子驱动FimA基因,以CTB为黏膜免疫佐剂的高效植物表达载体。AIM：To construct a plant effective expression vector driven by a fruit specific promoter for the expression of FimA （ Porphyromonas gingivalis Fimbriae A） gene so as to provide the basis for the development of an effective anti-periodontitis vaccine. METHODS：Genomic DNA of tomato and Porphyromonas gingivalis Fimbriae A were extracted. The core sequence（ about 1. 11kb） of tomato fruit specific promoter was amplified,By SOE PCR （ gene splicing by overlap extension） ,Fim A gene and the core sequence were spliced to form the EF sequence. EF sequence and expression vector pCAMBIA1302 were digested with restriction enzymes EcoR I and PstI respectively,and then EF sequence was subcloned to vector pCAMBIA1302 to yield the plasmid pCAMBIA1302-EF. The pCAMBIA1302-EF was finally directly introduced into Agrobacterium tumefaciens strain GV3103. RESULTS：PCR and restriction enzymes digestion proved that the recombinant vector contained the inserts with expected length of E8 promoter and the target fragments. CONCLUSION：In this study,plant expression vector containing FimA gene driven by tomato fruit specific promoter has been constructed successfully.

关 键 词：牙龈卟啉菌 蕃茄果实特异启动子E8 FimA 霍乱毒素B亚基 

分 类 号：R780[医药卫生—口腔医学]