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

Objective This study aimed at constructing secretory eukaryotic expression vector ofKGPcdgene encoding whole amino acid residues of mature KGPcd fromPorphyromonas gingivalisand investigating the transcription and expression of recom- bined plasmid VR1020/KGPcdin mammalian cells.Methods Eukaryotic expression plasmid VR1020/KGPcdwas constructed by using molecular cloning methods. Then, the VR1020/KGPcdwas transfected into mammalian cell COS7 with Lipofectamine 2000 according to the manufacturer′s instruction. The transcription of VR1020/KGPcdwas assayed by reverse transcription polymerase chain reaction (RT-PCR). The expression product of VR1020/KGPcdwas analyzed by using indirect immunofluorescence. The protein secretion in cultural medium was detected by ELISAmethod.Results It proved that the VR1020/KGPcdcould be tran- scribed 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/KGPcdwas 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.