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

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.