Abstract—

Reporter vectors expressing of amino acid residues 16–96 and 16–85 from Neh2-domain of Nrf2 transcription factor fused to firefly luciferase have been constructed. Cell lines stably expressing the above reporter constructs as well as an earlier developed Neh2-luc fusion reporter have been generated using HeLa cells. Comparative analysis of all three reporter lines has been performed using a well-known Nrf2 activator, nordihydroguaiaretic acid. The reporter expressing the minimal fragment of Neh2-domain containing 16–85 aa sequence exhibits a 2-fold higher amplitude of activation compared to Neh2-luc reporter, and thus can be recommended for high throughput screening for Nrf2 activators. A likely reason for the improved response of the above reporter could be ubiquitination of N-terminal lysine residues (7, 10, 11) of luciferase protein adjacent to the ubiquitinated Neh2 sequence.

中文翻译：

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Neh2-Luc报道分子用于抗氧化剂程序活化剂筛选的优化

摘要—

构建了表达与萤火虫荧光素酶融合的Nrf2转录因子的Neh2结构域16-96和16-85位氨基酸残基的报告载体。使用HeLa细胞已经产生了稳定表达上述报道基因构建体以及较早开发的Neh2-luc融合报道基因的细胞系。使用众所周知的Nrf2活化剂去甲二氢愈创木酸进行了所有三个报告基因系的比较分析。与Neh2-luc报告基因相比，表达Neh2结构域最小片段（含16-85个氨基酸序列）的报告基因的活化幅度高2倍，因此可建议用于Nrf2激活因子的高通量筛选。上述报告基因反应改善的可能原因可能是N端赖氨酸残基的泛素化（7、10，