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目的:构建真核表达载体p4ARE-TK-GFP/Neo,并在HepG2细胞中进行表达。方法:PCR法扩增TK启动子克隆到pEGFP-N1上,构建表达载体pTK-GFP/Neo。人工合成4个ARE序列,经退火和磷酸化后插入pTK-GFP/Neo载体的TK启动子上游,构建由TK启动子启动以及上游有4个ARE调控的真核表达载体p4ARE-TK-GFP/Neo,将其转染入HepG2细胞株并筛选,进行稳定表达。结果:成功构建了真核表达载体p4ARE-TK-GFP/Neo,并建立HepG2细胞稳定表达株。结论:成功地构建了由TK启动子启动以及上游有4个抗氧化反应元件(ARE)重复序列调控的真核表达载体p4ARE-TK-GFP/Neo,并在HepG2细胞中稳定表达,为下一步研究ARE的调控作用奠定了基础。
Objective: To construct eukaryotic expression vector p4ARE-TK-GFP / Neo and express it in HepG2 cells. Methods: The TK promoter was amplified by PCR and cloned into pEGFP-N1 to construct the expression vector pTK-GFP / Neo. Four ARE sequences were synthesized by annealing and phosphorylation, and inserted into the upstream of the TK promoter of pTK-GFP / Neo vector to construct an eukaryotic expression vector p4ARE-TK-GFP / Neo, transfected into HepG2 cell line and screened for stable expression. Results: The eukaryotic expression vector p4ARE-TK-GFP / Neo was successfully constructed and the stable HepG2 cell line was established. CONCLUSION: The eukaryotic expression vector p4ARE-TK-GFP / Neo, which is initiated by TK promoter and has four upstream repeat regulatory elements, was successfully constructed and stably expressed in HepG2 cells, which is the next step Study of the regulatory role of ARE laid the foundation.