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目的:构建人E2F1基因原核表达质粒p GEX-KG-E2F1,并在大肠杆菌中诱导表达。随后验证纯化得到的E2F1蛋白可作为底物被甲基化转移酶修饰。方法:构建原核表达质粒p GEX-KG-E2F1,在大肠杆菌BL-21中经异丙基硫代半乳糖苷(IPTG)诱导表达,利用GST亲和层析法纯化表达的E2F1蛋白。随后将纯化的E2F1蛋白作为底物,组蛋白甲基化转移酶SET7/9作为酶进行体外同位素标记放射自显影实验,检测纯化的E2F1蛋白能否被甲基化。结果:酶切鉴定和测序结果证明成功构建了原核表达载体p GEX-KG-E2F1,SDS-PAGE检测结果证明实现了人E2F1基因在大肠杆菌中的可溶性表达,放射自显影证明纯化得到的E2F1蛋白可作为底物被甲基化转移酶SET7/9甲基化。结论:成功构建了转录因子E2F1体外甲基化体系,为筛选新的能甲基化E2F1的酶奠定基础。
Objective: To construct the prokaryotic expression plasmid pGEX-KG-E2F1 of human E2F1 gene and induce it in E.coli. Subsequently, it was verified that the purified E2F1 protein could be modified by methylating transferase as a substrate. Methods: The prokaryotic expression plasmid pGEX-KG-E2F1 was constructed and induced by isopropylthiogalactoside (IPTG) in Escherichia coli BL-21. The expressed E2F1 protein was purified by GST affinity chromatography. Then purified E2F1 protein as a substrate, histone methyltransferase SET7 / 9 as an enzyme in vitro isotope labeled autoradiography test to detect whether the purified E2F1 protein can be methylated. Results: The recombinant plasmid pGEX-KG-E2F1 was successfully constructed by restriction enzyme digestion and sequencing. The results of SDS-PAGE showed that the human E2F1 gene was expressed in Escherichia coli. Autoradiography proved that the purified E2F1 protein It can be methylated by methyltransferase SET7 / 9 as a substrate. Conclusion: The in vitro methylation system of transcription factor E2F1 was successfully constructed and laid the foundation for the screening of new E2F1 capable of methylating E2F1.