【作者】 王辉； 韩静贤； 牛志国； 刘威； 高彩； 宋向凤；

【Author】 WANG Hui;HAN Jingxian;NIU Zhiguo;LIU Wei;GAO Cai;SONG Xiangfeng;School of Laboratory Medicine, Xinxiang Medical University;Research Center for Immunology, Xinxiang Medical University;

【机构】 新乡医学院医学检验学院； 新乡医学院免疫学研究中心；

【摘要】 目的:探讨HTLV-1病毒Tax蛋白阳性T细胞TaxP中早期生长反应基因-1表达调控的机制。方法:构建不同长度的EGR-1调控序列报告基因,将其转染TaxP细胞,加入NF-κB抑制剂BAY 11-7082或同等体积的DMSO,24h后收集细胞检测荧光素酶活性;TaxP细胞上清中加入BAY 11-7082或同等体积的DMSO,24h后,免疫印迹检测EGR-1蛋白表达;将Tax及其突变体M22和M47分别转染293T细胞,24h后免疫印迹检测EGR-1蛋白表达。结果:成功构建了不同长度及突变体的EGR-1调控序列荧光素酶报告基因;荧光素酶报告基因检测显示,相对于E1、E2而言,E3、DelE、MutE的荧光素酶活性明显降低,加入BAY 11-7082后,E1、E2的荧光素酶活性明显降低(P<0.01),而E3、DelE、MutE无明显变化;免疫印迹检测显示,加入BAY 10-7082后,EGR-1蛋白表达明显降低;相对于野生型和M47,转染M22突变体后,293T细胞EGR-1蛋白表达明显降低。结论:NF-κB是Tax蛋白阳性T细胞中EGR-1表达调控的关键核因子。更多还原

【Abstract】 The objective is to explore the expression and regulation mechanism of early growth response gene-1 in the HTLV-1 virus Tax protein-positive T cells TaxP. Methods: Build EGR-1 reporter gene regulatory sequences of different length and tansfected them into Tax P cells, add NF-κB inhibitor BAY 11-7082 or DMSO of equivalent volume, then collect cells after 24 hours and test the luciferase activity; add BAY 11-7082 or DMSO of equivalent volume to TaxP supernatant and test the EGR-1 protein expression by Western blot after 24 hours; transfect the Tax and its mutants M22 and M47 into 293T cells and test the EGR-1 protein expression by Western blot after 24 hours. Results: We successfully constructed EGR-1 regulatory sequence luciferase reporter gene of different length and mutants; the assay of luciferase reporter gene indicated that the luciferase activities of E3, DelE and MutE significantly decreased relative to E1 and E2. After adding BAY 11-7082, the luciferase activities of E1 and E2 significantly decreased(P<0.01) while there was no significant change in E3, DelE and MutE. Similarly, Western blot analysis indicated that EGR-1 protein expression significantly decreased after adding BAY 10-7082. Relative to wild type tax and M47, EGR-1 protein expression of 293T cells decreased significantly after the transfection of M22 mutants. Conclusion: NF-κB is the key nuclear factor and regulation in EGR-1 protein expression of Tax-positive T cells.更多还原