弓形虫SAG1-GRA1复合基因真核表达重组质粒的构建与表达  

Construction and Expression of Eukaryotic Expression Recombinant Plasmid of SAG1-GRA1 Gene of Toxoplasma gondii

作　　者：綦廷娜 石畅 汪政勇 宗永丽 李金福 QI Tingna;SHI Chang;WANG Zhengyong;ZONG Yongli;LI Jinfu(Department of Microbiology,Guizhou Medical University,Guiyang 550004,Guizhou,China;Department of Neuroendokrine,the 968 th Hospital of the People's Liberation Army of China,Jinzhou 121000,Liaoning,China;Department of Parasitology,Guizhou Medical University,Guiyang 550004,Guizhou,China)

机构地区：[1]贵州医科大学基础医学院微生物学教研室,贵州贵阳550004 [2]中国人民解放军联勤保障部队第968医院神经内分泌科,辽宁锦州121000 [3]贵州医科大学基础医学院寄生虫学教研室,贵州贵阳550004

出　　处：《贵州医科大学学报》2020年第3期286-291,共6页Journal of Guizhou Medical University

基　　金：贵州省科技厅科技支撑计划项目[黔科合SY字(2010)3145];贵州省科教青年英才培养工程项目[黔省专合字(2012)163];贵州省2016年大学生创新创业训练计划项目(201610660020)。

摘　　要：目的:构建弓形虫SAG1-GRA1复合基因真核表达重组质粒,并观察其在小鼠胚胎成纤维细胞系NIH3T3细胞中的表达。方法:采用聚合酶链反应(PCR)法分别扩增弓形虫SAG1和GRA1基因片段,导入质粒载体pc DNA3.1(+),构建真核表达重组质粒pc DNA3.1(+)-SAG1和pc DNA3.1(+)-GRA1,继而构建重组质粒pc DNA3.1(+)-SAG1-GRA1;设pc DNA3.1(+)-SAG1-GRA1为实验组、pc DNA3.1(+)为对照组分别转染NIH3T3细胞,采用免疫荧光染色法鉴定其在NIH3T3细胞中的表达。结果:限制性内切酶双酶切及DNA测序分析结果显示,插入pc DNA3.1(+)的片段分别为1008 bp、570 bp和1578 bp,与预期的SAG1、GRA1和SAG1-GRA1复合基因分子大小相当、序列一致;免疫荧光染色法结果显示,转染重组质粒pc DNA3.1(+)-SAG1-GRA1的NIH3T3细胞内观察到较强的绿色荧光,而转染pc DNA3.1(+)的NIH3T3细胞内未见绿色荧光。结论:成功构建了弓形虫SAG1-GRA1复合基因真核表达重组质粒pc DNA3.1(+)-SAG1-GRA1,且该重组质粒携带的SAG1-GRA1复合基因在NIH3T3细胞中获得表达。Objective:To construct eukaryotic expression recombinant plasmid of SAG 1-GRA 1 genes of Toxoplasma gondii and detect the gene expression of NIH3T3 cells.Methods:SAG 1 and GRA 1 genes were amplified from nuclear DNA of T.gondii isolates and cloned into an eukaryotic expression vector pcDNA3.1(+).The recombinant plasmid was named pcDNA3.1(+)-SAG 1 and pcDNA3.1(+)-GRA 1.Based on pcDNA3.1(+)-SAG 1 and pcDNA3.1(+)-GRA 1,pcDNA3.1(+)-SAG 1-GRA 1 was constructed.NIH3T3 cells were transfected with pcDNA3.1(+)-SAG 1-GRA 1 and pcDNA3.1(+)separately.The Expression of SAG 1-GRA 1 gene were detected by immunofluorescence.Results:The recombinant plasmid pcDNA3.1(+)-SAG 1,pcDNA3.1(+)-GRA 1 and pcDNA3.1(+)-SAG 1-GRA 1 were analyzed by DNA sequencing.The sizes of inserted fragments in the recombinant vectors were 1008 bp,570 bp and 1578 bp separately,corresponding to the gene molecules of SAG1,GRA1 and SAG 1-GRA 1.It was found that there was high green fluorescence inside the NIH3T3 cells transfected with pcDNA3.1(+)-SAG 1-GRA 1,but there was no green fluorescence inside the NIH3T3 cells transfected with pcDNA3.1(+).Conclusion:The eukaryotic expression recombinant plasmid of SAG 1-GRA 1 gene of T.gondii is constructed successfully and can be expressed in the NIH3T3 cells.

关 键 词：弓形虫属 SAG1基因 GRA1基因 基因重组 重组质粒 体外表达 

分 类 号：R382.5[医药卫生—医学寄生虫学]