【摘要】 目的:构建编码弓形虫RH株三磷酸核苷水解酶(NTPase-Ⅱ)重组真核表达载体PVAX1-NTPase-Ⅱ。方法:采用PCR从弓形虫基因组DNA中扩增NTPase-Ⅱ基因,克隆入pGEM-T Easy载体,并对重组入外源基因的质粒通过PCR、酶切和测序鉴定;采用亚克隆将NTPase-Ⅱ基因克隆至真核表达载体PVAX1,筛选阳性重组质粒PVAX1-NTPase-Ⅱ,进行PCR、酶切和测序鉴定。结果:NTPase-Ⅱ的重组真核表达载体经PCR、酶切和测序鉴定,大小为1 887 bp,与预期大小一致;重组真核表达载体的核苷酸序列,与GenBank中的相应序列100%同源。结论:成功构建重组真核表达载体PVAX1-NTPase-Ⅱ,为弓形虫核酸疫苗的研制奠定了基础。更多还原

【Abstract】 Objective: To construct an eukaryotic expression recombinant plasmid PVAX1-NTPase-Ⅱ.Methods: One gene fragments were amplified by PCR with primers that were designed according to the published gene sequence of NTPase-Ⅱ from Toxoplasma gondii RH strain Identified and then cloned into pGEM-T Easy vector,then subcloned into PVAX1 to generate eukaryotic expression plasmid PVAX1-NTPase-Ⅱ,were selected and identified by PCR,enzyme digestion and DNA sequencing analysis.Results: PCR,enzyme digestion and DNA sequencing analysis of PVAX1-NTPase-Ⅱ showed the length of fragment was 1 887 bp,which fitted the length of NTPase-Ⅱ.The sequence analysis demonstrated that the sequence identities were 100% between recombinant NTPase-Ⅱ gene and that from GenBank.Conclusion: The recombinant eukaryotic expression plasmid of PVAX1-NTPase-Ⅱhas been successfully obtained,building a foundation for the further studies on the DNA vaccine development for Toxoplasma gondii infection.更多还原