弓形虫表面抗原SAG1基因自杀性DNA疫苗的构建及免疫原性检测  

Construction and immunogenicity of a suicidal DNA vaccine with surface antigen SAG1 gene of Toxoplasma gondii

作　　者：郑丽娜[1,2] 谭峰[1] 潘长旺[1] 

机构地区：[1]温州医科大学基础医学院寄生虫学教研室,温州325035 [2]温州医科大学口腔医学院/附属口腔医院,温州325035

出　　处：《中国人兽共患病学报》2016年第10期885-888,892,共5页Chinese Journal of Zoonoses

基　　金：浙江省科技厅公益技术研究社会发展项目(No.2014C33161);温州市科技局公益项目(No.Y20140665);浙江省大学生科技创新活动计划暨新苗人才计划项目(No.2015R413061)联合资助~~

摘　　要：目的构建表达弓形虫表面抗原SAG1的自杀性DNA疫苗pDREP-SAG1并检测其免疫原性。方法以弓形虫RH株基因组DNA为模板,PCR扩增SAG1基因(GenBank No.HM776940.1),克隆至甲病毒复制子载体pDREP-eGFP中替换原有的eGFP基因,菌液PCR、双酶切及测序鉴定重组质粒。重组的自杀性DNA疫苗pDREP-SAG1经股四头肌注射并联合瞬时电穿孔免疫BALB/c小鼠,同时以原质粒pDREP-eGFP作为对照组,间隔3周后以相同条件再免疫1次,收集小鼠血清,以弓形虫裂解抗原Western blotting检测其诱导产生的特异性抗体。结果从弓形虫基因组中PCR扩增得到1 011bp长度的SAG1基因,成功构建重组质粒pDREP-SAG1。Western blotting结果显示,重组质粒免疫的小鼠血清能特异性地识别虫体抗原中的SAG1抗原。结论成功构建了弓形虫自杀性DNA疫苗pDREP-SAG1,能诱导小鼠产生特异性的抗体,具有免疫原性。We constructed a suicidal DNA vaccine pDREP-SAG1 encoding the major surface antigen SAG1 gene of Toxoplasma gondii and identify its immunogenicity. SAG1 gene （GenBank accession number： HM776940.1） fragment was amplified with PCR from genomic DNA of T. gondii RH strain. The PCR product was cloned into an alphavirus replicon-based vector pDREP-eGFP to replace the coding sequence of eGFP. Recombinant plasmid pDREP-SAG1 was confirmed by PCR, restriction enzymes digestion and sequencing. BALB/c mice were injected with the recombinant suicidal DNA vaccine pDREP-SAG1 intramuscularly into quadricep followed by immediate electroporation and boosted with the same condition once at 3-week intervals. Mice received injection with plasmid pDREP-eGFP were used as control. Sera collected from immunized mice were veri- fied by Western blotting with Toxoplasma lysate antigen （TLA）. Results showed that SAG1 gene of 1 011 bp was amplified from genomic DNA of T. gondii, and recombinant plasmid pDREP-SAG1 was successfully constructed. Sera collected from plasmid pDREP-SAG1 immunized mice could specifically recognize SAG1 antigen from TLA. The suicidal DNA vaccine pDREP-SAG1 was successfully constructed and showed its immunogenicity to induce specific antibody response in mice.

关 键 词：弓形虫 SAG1 自杀性DNA疫苗 

分 类 号：R382.5[医药卫生—医学寄生虫学]