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目的构建弓形虫抗原基因真核表达质粒pVAX1-SAG1和pVAX1-SAG4。方法根据弓形虫RH标准株SAG1和SAG4基因序列分别设计一对特异引物(分别含有HindⅢ/BamHⅠ和BamHⅠ/XbaⅠ内切酶位点),PCR扩增目的基因,并将这两段基因分别克隆至PEGM-T Easy载体,经菌落PCR和双酶切鉴定;用HindⅢ/BamHⅠ和BamHⅠ/XbaⅠ分别双酶切PEGM-T Easy-SAG1、PEGM-T Easy-SAG4和pVAX1,得到含内切酶位点的SAG1和SAG4基因片段,分别与pVAX1连接,构建pVAX1-SAG1和pVAX1-SAG4真核表达质粒,经菌落PCR和双酶切鉴定。结果PCR扩增得到目的基因SAG1和SAG4,测序结果分别与弓形虫RH标准株SAG1和SAG4基因比较,符合率均为100%。构建PEGM-T Easy-SAG1、PEGM-T Easy-SAG4克隆质粒和pVAX1-SAG1、pVAX1-SAG4真核表达质粒,分别经菌落PCR和双酶切鉴定,显示722bp的SAG1基因片段和511bp的SAG4基因片段均插入载体PEGM-T Easy和pVAX1中。结论成功克隆了pVAX1-SAG1、pVAX1-SAG4真核表达质粒,为弓形虫基因疫苗应用研究奠定了基础。
Objective To construct the eukaryotic expression plasmids pVAX1-SAG1 and pVAX1-SAG4 of Toxoplasma gondii antigen. Methods According to the sequence of SAG1 and SAG4 of RH strain Toxoplasma gondii, a pair of specific primers (containing Hind Ⅲ / BamH Ⅰ and BamH Ⅰ / Xba Ⅰ endonuclease sites respectively) were designed. The target gene was amplified by PCR and cloned into PEGM-T Easy vector, identified by colony PCR and double enzyme digestion. Double enzyme digestion of PEGM-T Easy-SAG1, PEGM-T Easy-SAG4 and pVAX1 with HindⅢ / BamHⅠ and BamHⅠ / The SAG1 and SAG4 gene fragments were respectively ligated with pVAX1 to construct eukaryotic expression plasmids of pVAX1-SAG1 and pVAX1-SAG4 and identified by colony PCR and double enzyme digestion. Results The target genes SAG1 and SAG4 were amplified by PCR. The sequencing results were compared with the SAG1 and SAG4 genes respectively. The coincidence rates were 100%. The eukaryotic expression plasmids of PEGM-T Easy-SAG1, PEGM-T Easy-SAG4 and pVAX1-SAG1, pVAX1-SAG4 were constructed and identified respectively by colony PCR and double enzyme digestion. The SAG1 gene fragment of 722bp and the SAG4 gene of 511bp The fragments were inserted into the vectors PEGM-T Easy and pVAX1. Conclusion The eukaryotic expression plasmids pVAX1-SAG1 and pVAX1-SAG4 were successfully cloned, which laid the foundation for the application of the DNA vaccine against Toxoplasma gondii.