弓形虫三磷酸核苷水解酶基因真核表达载体的构建

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1887 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.%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 PVAXl-NTPase-II, were selected and identified by PCR, enzyme digestion and DNA sequencing analysis. Results: PCR, enzyme digestion and DNA sequencing analysis of PVAXl-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 PVAXl-NTPase-II has been successfully obtained, building a foundation for the further studies on the DNA vaccine development for Toxoplasma gondii infection.