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目的构建刚地弓形虫(Toxoplasma gondii)棒状体蛋白11(ROP11)的真核表达重组质粒并在真核细胞中表达目的蛋白。方法设计合成弓形虫ROP11基因引物,运用RT-PCR方法扩增ROP11基因并克隆至真核表达质粒pcDNA3.1(+),构建重组表达质粒pcDNA3.1-ROP11。将重组质粒转染HeLa细胞,采用间接免疫荧光法检测目的蛋白表达。结果 RT-PCR扩增弓形虫ROP11基因片段为1 548bp,与预期大小相符。构建的重组质粒pcDNA3.1-ROP11经PCR及EcoRⅠ和NotⅠ双酶切鉴定正确。重组质粒测序后与GenBank报道的ROP11基因比对,核苷酸序列同源性和推导氨基酸序列同源性均为99%。免疫荧光检测显示,在重组质粒转染的HeLa细胞胞浆观察到黄绿色荧光,对照组无荧光。结论成功构建了真核表达质粒pcDNA3.1-ROP11,该重组质粒能够在真核细胞中表达目的蛋白,为弓形虫核酸疫苗的研制奠定了基础。
Objective To construct eukaryotic expression recombinant plasmid of Toxoplasma gondii rod protein 11 (ROP11) and express the target protein in eukaryotic cells. Methods The primers of ROP11 gene were designed and synthesized. The ROP11 gene was amplified by RT-PCR and cloned into eukaryotic expression plasmid pcDNA3.1 (+) to construct recombinant expression plasmid pcDNA3.1-ROP11. The recombinant plasmid was transfected into HeLa cells and the expression of the target protein was detected by indirect immunofluorescence. Results RT-PCR amplified Toxoplasma gondii ROP11 gene fragment of 1 548bp, with the expected size. The constructed recombinant plasmid pcDNA3.1-ROP11 by PCR and EcoR Ⅰ and Not Ⅰ double digestion correct identification. The recombinant plasmid was sequenced and compared with the ROP11 gene reported in GenBank. The nucleotide sequence homology and deduced amino acid sequence homology were both 99%. Immunofluorescence showed that yellow-green fluorescence was observed in the cytoplasm of HeLa cells transfected with the recombinant plasmid and no fluorescence in the control group. Conclusion The eukaryotic expression plasmid pcDNA3.1-ROP11 was constructed successfully. The recombinant plasmid can express the target protein in eukaryotic cells and lay a foundation for the development of Toxoplasma nucleic acid vaccine.