中文摘要：

目的构建可条件性敲除约氏疟原虫ebl基因的打靶载体。方法以约氏疟原虫基因组DNA为模板，PCR扩增EBL-3u和EBL-5U1、EBL-5U2＋EBL．off同源臂片段，利用MuhiSiteGateway技术，完成若干DNA片段的定性重组，构建打靶载体pCHD-EBL-RFT，并对质粒酶切和测序鉴定。PCR扩增约氏疟原虫UIS4—5U片段，置换质粒PbUIS4／flp中原有同源臂，并通过位点特异突变技术，获取AvrlI和NheI两种线性化位点，构建插入载体PyUIS4／flp。结果构建出打靶载体pCHD—EBL—FRT和PyUIS4／flp，酶切鉴定和测序分析正确。结论将MuhiSiteGateway技术成功地用于疟原虫条件打靶载体的构建，建立了以MuhiSiteGateway技术为基础的打靶载体构建体系。

英文摘要：

Objective To construct targeting vector for conditional knockout of ebl gene in Plasmodium yoelii using the Gateway recombi- natorial cloning system. Methods Three fragments EBL-3U,EBL-5U1 and EBL-5U2＋EBL.orf were amplified from Plasmodium yoelii 17xl strain by PCR. In order to get plasmid pCHD-EBL-FRT, multiple DNA fragments were ligated in a defined order and orientation using Mul- tiSite Gateway Technology. To get plasmid PyUIS4/flp, the PyUIS4-5U fragment was amplified by polymerase chain reaction by using genome from Plasmodium yoelii 17xl strain and replaced the homologous tuna of PbUIS4-5U fragment of the plasmid PbUIS4/flp. The plasmids were verified by restriction endonucleases digestion and sequencing. Results The vectors pCHD-EBL-FRT and PyUIS4/flp were correctly con- structed, which was confirmed by enzyme digestion and DNA sequencing. Conclusion The targeting vectors were successfully constructed by using MultiSite Gateway technology. A rapid and high efficient working system based on MultiSite Gateway technology was established for plasmodium gene-targeting vector construction.