BTB-Kelch蛋白KLHL32编码基因的克隆及初步鉴定

IM: To identify and characterize human KLHL32 gene, a member of BTB-Kelch family. METHODS: Real-time PCR was applied to analyze the expression of KLHL32 gene in PMA-induced THP-1 macrophage like cells. The full length of human KLHL32 coding region was amplified by RT-PCR and the amplicon was subcloned into eukaryotic expression plasmid pcDNA3.1. The effects of overexpression of KLHL32 on TNFα induced activation of NF-κB was analyzed with dual-luciferase reporter assay system. And the interaction between KLHL32 and Cul-3 was analyzed by co-immunoprecipitation assay. RESULTS: The expression of KLHL32 was downregulated in PMA-induced THP-1 macrophage cells upon 100ng/ml ST-FLA treatment. Human KLHL32 has been successfully cloned and subcloned into eukaryotic expression plasmid to get recombinant plasmid pcDNA3.1-KLHL32-FLAG. Overexpresion of KLHL32 in HEK293 cells didn't interfere the activity of transcription factor NF-κB activated by TNFα treatment. And KLHL32 interacts with Cul-3 protein through its BTB domain. CONCLUSION: Human KLHL32 gene has been identified for the first time. It can interact with Cul-3 through its BTB domain and may function as a potential E3 ubiquitin ligase in cells. The activation of TLR5 receptor leads to the downregulation of KLHL32 which suggests that KLHL32 may play a role in the cellular inflammatory response.