双顺反子表达载体DV的构建及其表达效率

Construction and Expression Efficiency of Bicistronic Expression Vector DV

目的构建双顺反子表达载体DV,并检测其表达效率。方法以载体VR1012为模板,PCR扩增BGH基因和启动子CMV-intronA基因片段,分别连接至载体VR1012启动子和终止子之间的相应位点上,构建双顺反子表达载体DV。另以质粒pshuttle-luciferase为模板,PCR扩增报告基因luciferase片段,分别连接至DV的两个启动子后,得到质粒DV-luc1和DV-luc2。将质粒DV-luc1、DV-luc2和阳性质粒pshuttle-luciferase分别转染COS-7细胞,Western blot检测luciferase蛋白的表达情况,荧光酶标仪检测luciferase降解底物的水平。结果双顺反子表达载体DV经双酶切鉴定证明构建正确;转染重组质粒DV-luc1和DV-luc2的COS-7细胞中均有luciferase的表达,且表达效率DV-luc2略高于DV-luc1。结论 已成功构建了双顺反子表达载体DV,且载体的第2个启动子的启动效率略高于第1个启动子,为基因治疗、转录调控、多价疫苗等的研究奠定了基础。

Objective To construct a bicistronic expression vector DV and determine its expression efficiency.Methods BGH gene and promoter CMV-intronA gene fragments were amplified by PCR using vector VR1012 as a template,and inserted into the corresponding sites between promoter and terminator of vector VR1012 to construct bicistronic expression vector DV.Luciferase gene fragment as a report gene was amplified by PCR using plasmid pshuttle-luciferase as a template and cloned into vector DV behind the two promoters respectively to obtain new vectors DV-luc1 and DV-luc2.COS-7 cells were transfected with DV-luc1,DV-luc2 and pshuttle-luciferase respectively,then determined for the expression levels of luciferase by Western blot and the ability of luciferase in substrate degradation by enzyme-labeled fluorescent apparatus.Results Restriction analysis proved that bicistronic expression vector DV was constructed correctly.Luciferase was expressed in both the COS-7 cells transfected with DV-luc1 and DV-luc2,while the expression efficiencies in the latter was slightly higher than that in the former.Conclusion Bicistronic expression vector DV was successfully constructed,of which the priming efficiency of promoter 2 was slightly higher than that of promoter 1.It laid a foundtion of studies on gene therapy,transcriptional regulation and multivalent vaccine.