蓝氏贾第鞭毛虫稳定表达载体的构建

Construction of a stable expression vector of Giardia lamblia

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 of Giardia lamblia. Methods To obtain the recombinant vec- tor pGL tim gdh-Neo for integration into the Giardia genome, the tim gene and its flanking sequence were inserted upstream of the 5＂UTR of gdh to yield pGI. gdh-Neo, as was constructed previously. A DNA fragment containing the 5＂UTR of a2-Tubulin, 16 multiple cloning sites （MCS）, and the 3＂UTR was synthesized and cloned into the pGL tim-gdh Neo using ApaI to construct the recombinant vector pGL tim-Tub-MCS^gdh-Neo. To validate the effectivity of the recombinant vector pGL tim-Tub-MCS- gdh-Neo, the Luc coding sequences were amplified by PCR using pTAL-Luc as a template. The purified DNA fragment was in- serted into MCS of pGL tim-Tub-MCS-gdh-Neo to generate the Luc expression vector pGL tim-Tub-Luc^gdh-Neo. The resulting plasmid was linearized and transfected into Cffardia trophozoites. The Luc recombinant strain was selected using G418 and its lu- ciferase activity was tested. Results A stable expression recombinant vector 5,395 bp in length was constructed with multiple cloning sites and the Neo selection marker. The Luc expression vector was transfected into Giardia trophozoites and its luciferase activity was markedly higher than the control. Conclusion A stable expression recombinant vector of （3. lamblia, pGL tim- Tub-MCS-gdh-Neo, was constructed.