牙龈卟啉单胞菌基因真核表达载体构建及其在哺乳动物细胞中的表达  

Construction of Eukaryotic Expression Vector for KGPcd Gene from Porphyromonas gingivalis and Expression in Mammalian Cells

作　　者：张凤秋 杨连甲 吴织芬 秦鸿雁[3] 

机构地区：[1]第四军医大学口腔医院牙周科 [2]第四军医大学口腔医院病理科 [3]第四军医大学医学遗传学与发育生物学教研室,陕西西安710032

出　　处：《华西口腔医学杂志》2004年第6期503-506,共4页West China Journal of Stomatology

摘　　要：目的　构建分泌型牙龈卟啉单胞菌牙龈蛋白酶K基因的真核表达载体VR10 2 0 KGPcd ,并检测其在哺乳动物细胞中的表达及分泌情况。方法　应用基因重组方法 ,构建真核表达载体VR10 2 0 KGPcd。然后用Lipofectamine2 0 0 0介导瞬时转染COS7细胞 ,以反转录聚合酶链反应 (RT_PCR)和间接免疫荧光检测重组质粒的基因转录和蛋白表达 ,酶联免疫吸附实验 (ELISA)检测培养上清中蛋白分泌情况。结果　转染的COS7细胞中可检测到目的基因的转录和表达 ,并且在培养的上清中检测到其表达的蛋白质。结论　成功构建了可分泌表达的真核表达载体VR10 2 0 KGPcd ,并在哺乳动物细胞中能够正确转录和翻译 ,培养上清中可检测到正确表达的目的蛋白 ,这为其作为基因疫苗免疫动物奠定了基础。Objective This study aimed at constructing secretory eukaryotic expression vector of KGPcd gene encoding whole amino acid residues of mature KGPcd from Porphyromonas gingivalis and investigating the transcription and expression of recombined plasmid VR1020/KGPcd in mammalian cells.Methods Eukaryotic expression plasmid VR1020/KGPcd was constructed by using molecular cloning methods. Then, the VR1020/KGPcd was transfected into mammalian cell COS7 with Lipofectamine 2000 according to the manufacturer′s instruction. The transcription of VR1020/KGPcd was assayed by reverse transcription polymerase chain reaction (RT-PCR). The expression product of VR1020/KGPcd was analyzed by using indirect immunofluorescence. The protein secretion in cultural medium was detected by ELISA method.Results It proved that the VR1020/KGPcd could be transcribed and translated into transfected COS7 cells. The expressed targeted protein could be secreted into cultural supernatant and could be detected by ELISA.Conclusion The eukaryotic expression plasmid of VR1020/KGPcd was constructed successfully and its product can be expressed in mammalian cells. The results indicated that the recombinant plasmid has antigenicity and may be acted as candidate gene vaccine. This laid a basis for its use as gene vaccine candidates in the development of anti-periodontitis and paved the way for further study.

关 键 词：牙周炎 牙龈卟啉单胞菌 牙龈蛋白酶K 基因疫苗 

分 类 号：R780.2[医药卫生—口腔医学]