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目的构建上游刺激因子2(USF2)及其截短体的真核表达载体,鉴定USF2蛋白中抑制泛素连接酶Smurf1/2转录的功能区域。方法采用PCR技术扩增USF2及其两个截短体USF2(1~235aa)、USF2(236~346aa)编码基因,分别将其构建在pCMV-Myc重组载体上,转染真核细胞后验证其表达,Western blot及实时定量PCR确定USF2下调Smurf1/2转录水平的区域。结果成功地将USF2及其两个截短体编码基因构建至pCMV-Myc载体,并验证其在真核细胞中的正确表达,Western Blot和实时定量PCR实验结果显示,USF2 C端236~346aa区域对Smurf1/2的转录水平有抑制效应。结论 USF2通过其C端236~346aa区域对Smurf1/2的转录产生抑制效应。
Objective To construct the eukaryotic expression vector of upstream stimulation factor 2 (USF2) and its truncated truncated fragment to identify the functional region of USF2 protein that inhibits ubiquitin ligase Smurf1 / 2 transcription. Methods The gene encoding USF2 and its truncated USF2 (1 ~ 235aa) and USF2 (236 ~ 346aa) was amplified by PCR. The recombinant plasmid was constructed on the pCMV-Myc recombinant vector and transfected into eukaryotic cells Expression, Western blot and real-time quantitative PCR were used to determine the region where USF2 down-regulated the transcription level of Smurf1 / 2. Results USF2 and its two truncated genes were successfully constructed into pCMV-Myc vector and verified in eukaryotic cells. Western Blot and real-time PCR results showed that the region of 236 ~ 346aa at the C terminus of USF2 Inhibition of Smurf1 / 2 transcriptional level. Conclusion USF2 has an inhibitory effect on the transcription of Smurf1 / 2 via its C terminal 236 ~ 346aa region.