CAJ | 학술논문

目的：构建一种携带转录蛋白AP2α效应元件的荧光素酶报告基因载体，并应用于筛选BMPs对AP2α转录活性的影响。方法设计4个串联的AP2α效应元件(AP2α-RE)，将其克隆至经Bam HⅠ和MluⅠ双酶切的pBGLuc荧光素酶报告基因质粒构建pBGLuc-AP2α-RE载体。重组腺病毒Ad-AP2α及其两种显性负性突变体Ad-dnAP2α感染小鼠间充质干细胞C3H10，通过Real-time PCR、Western blot检测AP2α的mRNA和蛋白水平表达，EMSA检测AP2α的DNA结合能力。pBGLuc-AP2α-RE转染C3H10细胞，荧光素酶报告基因检测同上各组AP2α的转录活性。Ad-BMPs感染pBGLuc-AP2α-RE转染的C3H10细胞，荧光素酶报告基因筛选BMPs对AP2α转录活性的影响。结果经PCR、酶切及测序鉴定，证实AP2α-RE正确克隆至pBGLuc-AP2α-RE荧光素酶报告基因载体，Ad-AP2α感染能显著提高AP2α的表达及结合DNA的能力。显性负性突变体可以表达各自的突变体， EMSA结果显示Ad-dnAP2α-△bHLH组能显著抑制AP2α结合DNA的能力，而Ad-dnAP2α-△TAD组结合的DNA探针强于对照组。荧光素酶报告基因提示AP2α过表达组能促进AP2α转录活性，而两种显性负性突变体均能抑制AP2α转录活性。重组腺病毒BMPs感染组的AP2α转录活性均有增高，其中BMP9最为显著。结论成功构建携带转录蛋白AP2α效应元件的荧光素酶报告基因载体用于检测AP2α的转录活性，并初步发现BMP9对AP2α的转录活性有明显的促进作用。
목적：구건일충휴대전록단백AP2α효응원건적형광소매보고기인재체，병응용우사선BMPs대AP2α전록활성적영향。방법설계4개천련적AP2α효응원건(AP2α-RE)，장기극륭지경Bam HⅠ화MluⅠ쌍매절적pBGLuc형광소매보고기인질립구건pBGLuc-AP2α-RE재체。중조선병독Ad-AP2α급기량충현성부성돌변체Ad-dnAP2α감염소서간충질간세포C3H10，통과Real-time PCR、Western blot검측AP2α적mRNA화단백수평표체，EMSA검측AP2α적DNA결합능력。pBGLuc-AP2α-RE전염C3H10세포，형광소매보고기인검측동상각조AP2α적전록활성。Ad-BMPs감염pBGLuc-AP2α-RE전염적C3H10세포，형광소매보고기인사선BMPs대AP2α전록활성적영향。결과경PCR、매절급측서감정，증실AP2α-RE정학극륭지pBGLuc-AP2α-RE형광소매보고기인재체，Ad-AP2α감염능현저제고AP2α적표체급결합DNA적능력。현성부성돌변체가이표체각자적돌변체， EMSA결과현시Ad-dnAP2α-△bHLH조능현저억제AP2α결합DNA적능력，이Ad-dnAP2α-△TAD조결합적DNA탐침강우대조조。형광소매보고기인제시AP2α과표체조능촉진AP2α전록활성，이량충현성부성돌변체균능억제AP2α전록활성。중조선병독BMPs감염조적AP2α전록활성균유증고，기중BMP9최위현저。결론성공구건휴대전록단백AP2α효응원건적형광소매보고기인재체용우검측AP2α적전록활성，병초보발현BMP9대AP2α적전록활성유명현적촉진작용。
Objective To construct a luciferase reporter vector containing the response element of transcription protein AP2αfor screening the effect of bone morphogenetic proteins (BMPs) on the transcriptional activity of AP2α. Methods Four tandem-linked response elements of AP2αwere cloned to the pBGLuc luciferase reporter gene plasmid, which was digested with Bam HI and Mlu I to construct pBGLuc-AP2α-RE vector. The recombinant adenovirus Ad-AP2αand its dominant negative mutant Ad-dnAP2α were used to infect mouse mesenchymal stem cells C3H10; the changes in cellular AP2α mRNA and protein expressions were detected by real-time PCR and Western blotting, and electrophoretic mobility shift assay (EMSA) was carried out to assess the DNA-binding ability of AP2α. C3H10 cells were transfected with pBGLuc-AP2α-RE vector, and AP2αtranscriptional activity was measured using luciferase reporter gene assay. In pBGLuc-AP2α-RE vector-transfected C3H10 cells infected with Ad-BMPs, luciferase reporter gene assay was performed to screen the effect of BMPs on AP2α transcriptional activity. Results The results of PCR, enzyme digestion and sequencing all confirmed correct cloning of AP2α-RE into pBGLuc-AP2α-RE luciferase reporter vector, and Ad-AP2α infection significantly increased AP2α expression and its DNA binding ability. The dominant negative mutants expressed the corresponding mutants, and EMSA results showed that Ad-dnAP2α-△bHLH significantly lowered while Ad-dnAP2α-△TAD enhanced the DNA-binding ability of AP2α. AP2α over-expression promoted AP2α transcriptional activity, which was suppressed by the two dominant negative mutants. AP2α transcriptional activity increased in the cells infected with the recombinant adenovirus BMPs, especially in cells with BMP9 infection. Conclusions The luciferase reporter vector containing the response element of AP2αwe constructed allows detection of AP2αtranscriptional activity. BMP9 can significantly enhance AP2αtranscriptional activity.