摘要
目的：克隆表达并研究cnot2（CCR4-NOT转录复合体亚基2）基因在哺乳动物生物钟信号通路中的作用，为进一步研究生物钟信号通路提供理论依据。方法提取B6小鼠的总RNA进行体外反转得到cDNA。依据小鼠的基因序列设计合成一对特异性引物，以反转录的小鼠cDNA为模板，扩增得到cnot2的基因片段。将基因克隆到表达载体，转染HEK293T细胞系，获得总蛋白后通过Western blot检测该基因在体外是否成功表达。然后设计实验体系，收集细胞利用报告基因检测试剂盒检测per1-luc的活性，最后通过免疫共沉淀技术证明cnot2与主要生物钟基因之间的相互作用。结果 cnot2基因能够抑制哺乳动物生物钟基因bmal1和clock的转录活性，其可能的机制是cnot2能够竞争性地结合bmal1和clock，使bmal1-clock复合体的转录活性降低。结论 cnot2基因是生物钟信号通路中的一个负向因子。
Objective To clone and investigate the role of the cnot2 gene in the circadian clock signal path, providing a theoretical basis for further study of the circadian clock signaling pathway. To extract RNA from the B6 mice in vitro, inverting it to cDNA. Based on mouse gene sequence to synthesis a pair of specifi c primers, then to amplify DNA fragment of the cnot2 gene by using mouse cDNA as a template. The fragment was cloned into the expression vector, and to detect whether the gene was successfully expressed in vitro, we transfect it to HEK293T cell line, obtaining the total protein and analysed by Western blot, then design experimental system and collect the cells by using Dual-Luciferase Reporter Assay System kit to detect the activity of the per1-luc, fi nally through co-precipitation technique to prove the interaction between cnot2 and the main clock gene.Result cnot2 could inhibit the transcriptional activity of clock and bmal1, which is mammalian circadian clock genes. The possible mechanism is that cnot2 could competitive bind to bmal1 and clock.Conclusion In circadian clock signaling pathway, cnot2 is a negative factor.
出处
《中国血液流变学杂志》
CAS
2015年第3期263-266,共4页
Chinese Journal of Hemorheology
