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目的:克隆青海湖裸鲤低氧诱导因子-2α(HIF-2α)完整编码区,并分析HIF-2αODD域与脯氨酸羟化酶3(PHD3)的相互作用。方法:通过RT-PCR与RACE克隆青海湖裸鲤HIF-2α基因序列,GST pull-down法分析HIF-2αODD域能否与PHD3相互结合。结果:获得的青海湖裸鲤HIF-2αcDNA序列长为3 013 bp,其中开放阅读框(ORF)为2 502 bp。GST pull-down分析表明,HIF-2αODD域羟化后能与PHD3形成酶/底物聚合物。结论:青海湖裸鲤HIF-2αODD域没有发生类似HIF-1α的羟化位点变异,能够被PHD3正常识别并结合。
OBJECTIVE: To clone the complete coding region of hypoxia-inducible factor-2α (HIF-2α) in naked carp of Qinghai Lake and analyze its interaction with proline hydroxylase 3 (PHD3). Methods: HIF-2α gene sequence was cloned by RT-PCR and RACE from Qinghai Lake. GST pull-down method was used to analyze whether HIF-2α ODD domain could bind to PHD3. Results: The sequence of HIF-2α cDNA of naked carp collected from Qinghai Lake was 3 013 bp, with an open reading frame (ORF) of 2 502 bp. GST pull-down analysis showed that the HIF-2 [alpha] ODD domain was hydroxylated to form an enzyme / substrate polymer with PHD3. CONCLUSION: HIF-2αODD domain in Qinghai Lake showed no HIF-1α-like hydroxylation site mutation, which could be recognized and combined by PHD3.