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目的 体外扩增弓形虫RH株致密颗粒蛋白GRA1基因 ,并构建真核表达质粒。方法 从接种了弓形虫RH株的小鼠腹水中收集、纯化速殖子 ,提取基因组DNA ;据已知的GRA1基因列 ,设计合成一对引物 ,并引入EcoRⅠ和BamHⅠ酶切位点。应用PCR技术 ,从弓形虫RH株基因组DNA中扩增GRA1基因片段。扩增目的基因片段经纯化、双酶切后 ,插入真核表达质粒pEGFP -N3中 ,转化大肠杆菌DH5α感受态细胞 ,于卡那霉素阳性LB平板上筛选阳性克隆。重组子经EcoRⅠ和BamHⅠ双酶切、PCR鉴定。结果 从弓形虫RH株基因组DNA中扩增出 785bp的GRA1基因片段 ,构建重组质粒pEGFPN3-GRA1,酶切和PCR鉴定产物大小均与预期值相符。 结论 成功地从弓形虫RH株基因组DNA中获取了GRA1基因 ,并构建了pEGFPN3-GRA1重组质粒。为重组质粒的进一步表达和核酸疫苗的研究创造了条件
Objective To amplify the GRA1 gene of Toxoplasma gondii RH strain in vitro and construct the eukaryotic expression plasmid. Methods Tachyzoites were collected and purified from ascites of mice inoculated with Toxoplasma gondii RH strain. Genomic DNA was extracted. According to the known GRA1 gene sequence, a pair of primers was designed and synthesized, and EcoR Ⅰ and BamH Ⅰ restriction sites were introduced. The GRA1 gene fragment was amplified from genomic DNA of Toxoplasma gondii RH strain by PCR. The target gene fragment was amplified, double digested and inserted into eukaryotic expression plasmid pEGFP-N3. The recombinant plasmid was transformed into E. coli DH5α competent cells, and the positive clones were screened on kanamycin-positive LB plates. The recombinant plasmid was digested with EcoRⅠ and BamHⅠ and identified by PCR. Results The 785bp GRA1 gene fragment was amplified from the genomic DNA of Toxoplasma gondii RH strain, and the recombinant plasmid pEGFPN3-GRA1 was constructed. The size of digested products and PCR products were consistent with the expected values. Conclusion The GRA1 gene was successfully obtained from the genomic DNA of RH strain Toxoplasma gondii and the recombinant plasmid pEGFPN3-GRA1 was constructed. This provided conditions for the further expression of recombinant plasmids and the study of nucleic acid vaccine