二价双启动子防龋DNA疫苗的构建及鉴定

Construction and verification of the dual-promoter plasmid pCN-SSISG as bivalent anti-caries DNA vaccine

目的:构建并鉴定具有在真、原核细胞中均可表达目的抗原并具备强免疫原性、针对致龋菌变形链球菌2种主要毒力因子的DNA疫苗。方法:利用PCR技术从载有变形链球菌gtfB基因的质粒中扩增葡聚糖结合区段(CBR)基因片断;将合成的组织纤溶酶原信号肽序列(tPA-SP)连接到GBR基因的上游;再将此基因(sGBR)定向克隆到载有sSBR基因的双启动子真核表达载体pCN-SSIE上,构建出pCN-SSISG;对重组质粒pCN-SSISG进行酶切图谱分析和DNA序列测定。结果:酶切图谱分析显示,PCR扩增的及插入到pCN-SSIE中的目的基因sGBR片断大小与预期相符; DNA序列测定结果确定:重组质粒pCN-SSISG开放性阅读框架和插入的信号肽序列均正确。结论:成功构建了具有在真核、原核宿主细胞均可表达的具有双启动子的质粒pCN-SSISC。

PURPOSE： The aim of the study is to construct an anti-caries DNA vaccine with high immunogenicity due to its unique properties of being able to express target antigens both in eukaryotic and prokaryotic host cells and harboring two main virulence genes from caries pathogen Streptoccocus mutans （S. mutans）. METHODS： The gene encoding glucan binding region （GBR） of glucosyhransferase-Ⅰ （GTF-Ⅰ） was amplified from the plasmid harboring the gtfB gene from S. mutans by PCR. Then the GBR gene that was upstream linked with tissue-type plasminogen activator signal peptide （tPA- SP） was directly cloned into the plasmid pCN-SSIE containing sSBR gene encoding tPA-SP and saliva binding region （SBR） of antigen protein Ⅰ/Ⅱ （AgⅠ/Ⅱ） from S. mutans. And finally the recombinant plasmid pCN-SSISG was analysed by DNA sequencing and endonuclearase digestion mapping. RESULTS： DNA sequencing and endonuclearase digestion mapping showed that the open reading frame （ORF） and the tPA-SP sequence of the recombinant plasmid pCN-SSISG were identical with the expectant ones. CONCLUSION： We have successfully constructed the dual-promoter bivalent recombinant plasmid pCN-SSISG.Supported by National Natural Science Foundation of China （Grant No.30371323, 30171010）.