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目的构建弓形虫核苷三磷酸水解酶-Ⅱ(NTPase-Ⅱ)基因真核表达质粒pcDNA3.1(+)-NTPase-Ⅱ并在COS-7细胞中进行瞬时表达。方法以pBAD-HisB-NTPase-Ⅱ质粒为模板,PCR扩增NTPase-Ⅱ目的基因,将其克隆到pcDNA3.1(+)真核表达载体中,双酶切及测序鉴定重组质粒。阳离子脂质体法转染COS-7细胞并经SDS-PAGE和Western Blot检测目的蛋白的表达。结果经鉴定,弓形虫pcDNA3.1(+)-NTPase-Ⅱ核酸疫苗质粒构建成功。以脂质体法转染COS-7细胞后,转染细胞可成功地表达弓形虫NTPase-Ⅱ蛋白。结论证实了弓形虫NTPase-Ⅱ蛋白能在真核细胞中表达,为该基因的核酸疫苗研究提供了实验依据。
Objective To construct the eukaryotic expression plasmid pcDNA3.1 (+) - NTPase-Ⅱ of Toxoplasma gondii NTPase-Ⅱ gene and express it transiently in COS-7 cells. Methods The target gene of NTPase-Ⅱ was amplified by PCR using pBAD-HisB-NTPase-Ⅱ as a template. The recombinant plasmid was cloned into eukaryotic expression vector pcDNA3.1 (+). COS-7 cells were transfected by cationic liposome method and the expression of the target protein was detected by SDS-PAGE and Western Blot. Results Toxoplasma gondii pcDNA3.1 (+) - NTPase-Ⅱ DNA vaccine plasmid was successfully constructed. After transfection with COS-7 cells by lipofectamine, the transfected cells could successfully express Toxoplasma gondii NTPase-Ⅱ protein. Conclusion The results showed that Toxoplasma gondii NTPase-Ⅱprotein was expressed in eukaryotic cells, which provided an experimental basis for the study of nucleic acid vaccine of this gene.