摘要
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 firefly luciferase expression vector driven by mouse WNT1 gene promoter and determine its expres- sion in P19 cell. Methods A fragment including the smallest functional unit of WNT1 gene promoter and 3＇UTR regions of the DNA was am- plified and inserted into pGL3 - Basic vector which contained a firefly luciferase reporter gene driven by WNT1 promoter. The recombinant vector or pGL3 - Basic vector was co - transfected together with pRL - cytomegalovirus （ CMV ） vector containing renila luciferase reporter gene into P19 cell. Firefly and renila luciferase activities were analyzed 24 h later by using of dual luciferase reporter assay system. Results Restriction enzyme double digestion and sequencing analysis confirmed correct construction of recombinant expression vector containing WNT1 promoter controlled firefly lueiferase reporter gene. The study compared the blank control group （ fluorescence intensity ratio ： 2. 820 4 ± 0. 294 4） with 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 ） , and the differences were statistically significant（ Pa 〈 0. 001 ） , which suggested that high expression of firefly luci- ferase was detected in P19 cell. Conclusions A firefly lueiferase expression vector driven by mouse WNT1 gene promoter can be successfully constructed,which specifically expresses firefly luciferase in P19 cell.
出处
《实用儿科临床杂志》
CAS
CSCD
北大核心
2012年第13期987-989,993,共4页
Journal of Applied Clinical Pediatrics
基金
国家自然科学基金(81070500)
