BTB-Kelch蛋白KLHL32编码基因的克隆及初步鉴定
王妮，陈新玉，欧小利，姜勇，梅柱中
基金项目：教育部新教师基金项目（20104433120009）； 国家自然科学基金 （81070955）
作者简介：王妮 （1988-），女，硕士研究生，炎症的信号转导
通信联系人：梅柱中（1973-），男，副教授，炎症的信号转导机制.

（南方医科大学基础医学院广东省蛋白质组学重点实验室，广东广州， 510515）
摘要：目的：克隆人BTB-Kelch家族蛋白KLHL32编码基因并初步鉴定其在细胞中的功能。 方法：采用荧光实时定量PCR分析TLR5特异性激活剂ST-FLA刺激经PMA诱导分化的THP-1巨噬细胞中KLHL32基因表达水平的变化，应用RT-PCR克隆了人KLHL32编码区cDNA序列并将其克隆至真核表达载体pcDNA3.1，采用荧光素酶双报告基因表达系统分析KLHL32过表达对转录因子NF-κB基因活性的影响，并采用免疫共沉淀实验分析了KLHL32与Cul-3蛋白在细胞内的相互结合。 结果：100ng/ml ST-FLA刺激4h诱导经PMA诱导分化的THP-1巨噬细胞中KLHL32基因表达水平下调，成功克隆了人KLHL32编码基因并将其克隆至真核表达载体中获得重组质粒pcDNA3.1-KLHL32-FLAG，在HEK293细胞中过表达KLHL32蛋白对TNFα诱导的转录因子NF-κB激活没有明显影响，但KLHL3215 可通过其氨基端的BTB结构域与Cul-3相互结合。结论：成功鉴定了人KLHL32蛋白的编码基因，其通过BTB结构域与Cul-3蛋白相互结合，可能是一种潜在的E3泛素连接酶，证实了TLR5受体激活可诱导其表达水平的下调，提示KLHL32蛋白在细胞中可能参与了炎症细胞信号转导通路的调控。
关键词：KLHL32； E3泛素连接酶；TLR5受体
中图分类号：Q786, R392.11
Cloning and preliminary characterization of KLHL32, a novel human gene encoding a BTB-Kelch protein
Wang Ni, Chen Xinyu, Ou Xiaoli, Jiang Yong, Mei Zhuzhong
(Key Laboratory of Functional Proteomics of Guangdong Province, Department of Pathophysiology, Southern Medical University, Southern Medical University，Guangzhou 510515，China)
Abstract: AIM: 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 30 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 35 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 40 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.
Key words: KLHL32，E3 ubiquitin ligase, TLR5 receptor