中文摘要：

目的：克隆人BTB-Kelch家族蛋白KLHL32编码基因并初步答定其在细胞中的功能。方法：采用实时荧光定量PCR分析Toll样受体5（Toll-likereceptor 5，TLR5）特异性激活剂鼠伤寒沙门菌鞭毛蛋白（flagllin from S．typhimurium．ST-FLA）刺激经佛波酯（phorbol 12-myristate 13-acetate，PMA）诱导分化的THP-1巨噬细胞中KLHL32 mRNA表达水平的变化，应用RT—PCR克隆人KLHId2编码区的cDNA序列并将其克隆至真核表达载体pcDNA3．1，采用萤光素酶双报告基因表达系统分析KLH132过表达对转录因子核测子κB（NF-κB）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：ST—FLA（100κg／L）刺激经PMA诱导分化的THP-1巨噬细胞4h引起KLHL32 mRNA的表达水平下调；成功克隆了人KLHL32基因并将其克隆至真核表达载体中获得最组质粒pcDNA3．1-KLH132-FLAG，存HEK293细胞中过表达KLHL32蛋白对TNF-α诱导的转录闪子NF-κB激活没有明显影响，但KLH132可通过其氨基端的BTB结构域与Cul-3相互结合。结论：成功签定了人KLHL32蛋白，其通过BTB结构域与Cul-3蛋F1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KLH132蛋白在细胞中可能参与炎症细胞信号转导的调控。

英文摘要：

AIM ： To identify and characterize the human KLHL32 protein, a member of BTB-Kelch family, in the regulation of inflammation. METHODS： Real-time PCR was applied to analyze the expression of KLHL32 mRNA in phorbol 12-myristate 13-acetate （PMA）-induced macrophage-like THP-1 cells stimulated hy flagellin from S. typhimurium （ ST-FLA）, an activator of Toll-like receptor 5 （ TLR5 ）. The full length of human KLHL32 coding region was amplified by RT-PCR, a.nd the amplicon was subcloned into a eukaryotic expression plasmid pcDNA3. 1. The effects of KLHL32 over- expression on TNF-α-induced activation of NF-κB was analyzed by dual-luciferase reporter assay. The protein interaction between KLHL32 and Cul-3 was determined by co-immunoprecipitation. RESULTS： The mRNA expression of KLHL32 was down-regulated in PMA-induced THP-1 cells upon 100 μg/L ST-FI,A treatment. Human KLHL32 was successfully chined anti subecloned into a eukaryotic expression plasmid to establish a recombinant plasmid peDNA3. 1-KLHL32-FLAG. Over-expression of KLHL32 in the HEK293 cells didn＇ t interfere the activity of transcription faetor NF-κB activated by TNF-α. KLH132 interacted with Cul-3 protein through its BTB domain. CONCLUSION： Human KLHL32 protein has been identified for the first time. It interacts with Cul-3 through its BTB domain and may function as a potential E3 ubiquitin ligase in the cells. The activation of TLR5 receptor leads to down-regulation of KLHL32, suggesting that KLHL32 may play a role in the cellular inflammatory response.