论文部分内容阅读
目的构建小鼠WNT1基因启动子控制的萤火虫荧光素酶表达载体,并检测其在P19细胞中的表达。方法采用PCR方法获得WNT1基因启动子最小功能单位和3’UTR区域在内的DNA片段。双酶切后插入到pGL3-Basic载体中构成重组表达载体,使萤火虫荧光素酶报告基因的表达受WNT1启动子控制。将构建的重组表达载体或pGL3-Basic载体分别与内参质粒pRL-SV40(表达海肾荧光素酶)共转染P19细胞,24 h后用双荧光检测试剂盒测定萤火虫荧光素酶及海肾荧光素酶活性。结果重组表达载体经双酶切及测序鉴定证明构建正确,空白对照组(荧光强度比值:2.820 4±0.294 4)与对照组(荧光强度比值:3.050 8±0.303 7)及WNT-3’UTR突变组(荧光强度比值:51.475 8±0.983 7)比较,差异均有统计学意义(Pa<0.001),该重组表达载体在P19细胞特异性高表达萤火虫荧光素酶。结论成功构建小鼠WNT1基因启动子控制的萤火虫荧光素酶表达载体,并检测到该载体在P19细胞的特异性表达。
Objective To construct a firefly luciferase expression vector controlled by mouse WNT1 promoter and detect its expression in P19 cells. Methods The DNA fragment containing the minimal functional unit of WNT1 promoter and 3’UTR region was obtained by PCR. Double digested and inserted into the pGL3-Basic vector to form a recombinant expression vector so that the firefly luciferase reporter gene expression is controlled by the WNT1 promoter. The constructed recombinant expression vector or pGL3-Basic vector was cotransfected into P19 cells with the plasmid pRL-SV40 (expressing Renilla luciferase), respectively, and after 24 h, firefly luciferase and Renilla luciferase Enzyme activity. Results The recombinant plasmid was confirmed by double enzyme digestion and sequencing. The results showed that the recombinant plasmid was constructed correctly, and the blank control group (fluorescence intensity ratio: 2.820 4 ± 0.294 4) and control group (fluorescence intensity ratio: 3.050 8 ± 0.303 7) and WNT-3’UTR mutation Group (fluorescence intensity ratio: 51.475 8 ± 0.983 7), the difference was statistically significant (Pa <0.001). The recombinant expression vector specifically expressed firefly luciferase in P19 cells. Conclusion The firefly luciferase expression vector controlled by the mouse WNT1 gene promoter was successfully constructed and the specific expression of this vector in P19 cells was detected.