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目的构建蓝氏贾第鞭毛虫基因的稳定表达载体。方法以本实验室构建的pGL gdh-Neo为基础,在gdh5′UTR端ApaⅠ酶切位点前插入蓝氏贾第鞭毛虫磷酸丙糖异构酶(tim)基因以方便载体与贾第虫基因组整合,得到pGLtim-gdh-Neo;设计含有16个酶切位点的多克隆位点区(MCS)并将其置于α2-Tubulin启动子调控下,使成为一个完整表达框,合成该表达框并单酶切插入重组质粒pGL tim-gdh-Neo的ApaⅠ位点,获得重组稳定表达质粒载体pGL tim-Tub-MCS-gdh-Neo。在MCS区插入Luc验证其有效性,以pTAL-Luc质粒为模板,扩增Luc基因序列,纯化后将其插入EcoRⅤ单酶切的pGL tim-Tub-MCS-gdh-Neo多克隆位点区,重组质粒线性化后转染虫体,并对G418筛选获得的Luc贾第虫重组株进行荧光素酶活性检测。结果构建了可稳定转染,带有多克隆位点区及Neo抗性基因的重组表达载体pGL tim-Tub-MCS-gdh-Neo,其长度为5 395bp;Luc重组质粒pGL tim-Tub-Luc-gdh-Neo可稳定转染至贾第虫并表达荧光素酶。结论成功构建了蓝氏贾第鞭毛虫稳定表达载体pGL tim-Tub-MCS-gdh-Neo。
Objective To construct a stable expression vector for Giardia lamblia. Methods Based on the pGL gdh-Neo constructed in our laboratory, the tim gene of Giardia lamblia was inserted in front of the ApaⅠ restriction site of gdh 5 ’UTR to facilitate the interaction between the vector and Giardia genome And then cloned into pGLtim-gdh-Neo to construct a polyclonal site (MCS) containing 16 restriction sites and placed under the regulation of α2-Tubulin promoter to make a complete expression cassette for synthesizing the expression cassette The recombinant plasmid pGL tim-Tub-MCS-gdh-Neo was successfully constructed and inserted into the Apa I site of the recombinant plasmid pGL tim-gdh-Neo. Luc was inserted into the MCS region to verify its effectiveness. The pTAL-Luc plasmid was used as a template to amplify the Luc gene sequence, then inserted into the EcoRV digested pGL tim-Tub-MCS-gdh-Neo multi-cloning site region, The recombinant plasmid was linearized and transfected into the parasite, and the luciferase activity of the recombinant Luc gicarci was detected by G418 screening. Results The recombinant plasmid pGL tim-Tub-MCS-gdh-Neo was constructed and transfected into pGL tim-Tub-Luc with a length of 5 395 bp. -gdh-Neo stably transfected Giardia and expressing luciferase. Conclusion The stable expression vector pGL tim-Tub-MCS-gdh-Neo was successfully constructed.