牙龈卟啉单胞菌HA2基因和白细胞介素IL-15基因重组质粒的构建

构建抗牙龈卟啉单胞菌的牙周炎基因疫苗p VAX1-HA2、pVAX1-HA2/IL-15,体外检测其在293T细胞的表达。以HA2基因(牙龈卟啉单胞菌牙龈素—血凝素基因编码区的核心功能区)为目的基因与IL-15基因为免疫佐剂构建真核表达质粒,用Lip2000介导瞬时转染293T细胞,RT-PCR检测目的基因mRNA水平及酶联免疫吸附试验检测IL-15蛋白表达水平。重组质粒p VAX1-HA2、pVAX1-HA2/IL-15经酶切及DNA测序鉴定构建正确,转染的293T细胞能够检测到目的基因的表达,也可以检测到IL-15蛋白的表达。说明我们成功构建了真核共表达质粒pVAX1-HA2和p VAX1-HA2/IL-15,为下一步研制抗牙龈卟啉单胞菌DNA疫苗奠定了基础。

The Study on the Construction and Expression of the Recombinant Plasmids of Porphyromonas gingivalis HA2 Gene and Interleukin IL-15 Gene

The periodontitis gene vaccines, pVAX1-HA2 and p VAX1-HA2/IL-15 against Porphyromonas gingivalis were constructed and their expression in 293 T cells was detected in vitro. Eukaryotic expression plasmid was constructed with HA2 gene(core functional region of gingipain-hemagglutinin gene coding region of Porphyromonas gingivalis) as target gene and IL-15 gene as immune adjuvants, Lip2000 mediated transient transfection of 293 T cells, RT-PCR was used to detect the mRNA level of target gene and enzyme-linked immunosorbent assay(ELISA) was used to detect the expression level of IL-15 protein. The recombinant plasmid pVAX1-HA2, pVAX1-HA2/IL-15 was constructed correctly by enzyme digestion and DNA sequencing, the transfected 293 T cells were able to detect the expression of the target gene, and the expression of IL-15 protein was also detected. These results indicated that eukaryotic co-expression plasmids pVAX1-HA2 and pVAX1-HA2/IL-15 were successfully constructed, which would lay the foundation for the further development of DNA vaccine against Porphyromonas gingivalis.