牙龈卟啉单胞菌FimA蛋白和IL-15真核共表达质粒的构建与鉴定

Objective To construct a eukaryotic co-expression plasmid pIRES-fimA:IL15, which can be used as an immunoreaction-enhancing DNA vaccine against Porphyromonas gingivalis FimA, and investigate its expression in mammalian cells. Methods The eukaryotic co-expression plasmid pIRES-fimA:IL15 was constructed by molecular cloning methods and characterized by restricted endonuclease mapping, PCR and DNA sequencing. The plasmid was transfected into mammalian cell CHO using Lipofectamine 2000. Expression of fimA gene was detected by Western blot and the protein secretion in cultural medium was analyzed by ELISA. Results Endonuclease mapping showed that the target genes fimA and IL-15 obtained by PCR had the same molecular size as predicted. The DNA sequencing data also indicated that inserted fimA gene and IL-15 gene had correct DNA sequence and orientation. The recombined plasmid could express FimA in mammalian cell CHO transfected. FimA and IL-15 could be secreted into cultural supernatant detected by ELISA. Conclusion A new eukaryotic co-expression plasmid pIRES-fimA:IL15 was constructed and it could be applied for further immunization in animal as an effective anti-Porphyromonas gingivalis vaccine.