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BTB-Kelch蛋白KLHL32編碼基因的克隆及初步鑒定
來源: | 作者: | 發布時間:2014-4-15 16:39:09

 BTB-Kelch蛋白KLHL32編碼基因的克隆及初步鑒定
王妮,陳新玉,歐小利,姜勇,梅柱中
基金項目:教育部新教師基金項目(20104433120009); 國家自然科學基金 (81070955)
作者簡介:王妮 (1988-),女,碩士研究生,炎癥的信號轉導
通信聯系人:梅柱中(1973-),男,副教授,炎癥的信號轉導機制.

(南方醫科大學基礎醫學院廣東省蛋白質組學重點實驗室,廣東廣州, 510515)
摘要:目的:克隆人BTB-Kelch家族蛋白KLHL32編碼基因并初步鑒定其在細胞中的功能。 方法:采用熒光實時定量PCR分析TLR5特異性激活劑ST-FLA刺激經PMA誘導分化的THP-1巨噬細胞中KLHL32基因表達水平的變化,應用RT-PCR克隆了人KLHL32編碼區cDNA序列并將其克隆至真核表達載體pcDNA3.1,采用熒光素酶雙報告基因表達系統分析KLHL32過表達對轉錄因子NF-κB基因活性的影響,并采用免疫共沉淀實驗分析了KLHL32與Cul-3蛋白在細胞內的相互結合。 結果:100ng/ml ST-FLA刺激4h誘導經PMA誘導分化的THP-1巨噬細胞中KLHL32基因表達水平下調,成功克隆了人KLHL32編碼基因并將其克隆至真核表達載體中獲得重組質粒pcDNA3.1-KLHL32-FLAG,在HEK293細胞中過表達KLHL32蛋白對TNFα誘導的轉錄因子NF-κB激活沒有明顯影響,但KLHL3215 可通過其氨基端的BTB結構域與Cul-3相互結合。結論:成功鑒定了人KLHL32蛋白的編碼基因,其通過BTB結構域與Cul-3蛋白相互結合,可能是一種潛在的E3泛素連接酶,證實了TLR5受體激活可誘導其表達水平的下調,提示KLHL32蛋白在細胞中可能參與了炎癥細胞信號轉導通路的調控。
關鍵詞:KLHL32; E3泛素連接酶;TLR5受體
中圖分類號: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

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