乙型肝炎病毒X蛋白截短突变体的构建及其对Wnt/β-catenin信号通路转录活性的影响  

Construction of hepatitis B virus X protein truncated mutants and their effects on β-catenin/TCF4 transcriptional activity in Wnt signaling pathway

作　　者：谢青[1] 陈林林[1] 李治[1] 单晓亮[1] 聂丹[1] 段玉洁[1] 权会琴[1] 唐霓[1] 

机构地区：[1]重庆医科大学感染性疾病分子生物学教育部重点实验室,重庆400016

出　　处：《中国病理生理杂志》2014年第1期35-40,共6页Chinese Journal of Pathophysiology

基　　金：国家自然科学基金资助项目(No.31171307;No.81371827);"十二五"传染病国家科技重大专项(No.2012ZX10002005-003-002;No.2013ZX10002002-005-003)

摘　　要：目的:观察乙型肝炎病毒X蛋白(HBx)截短突变体在细胞内的定位及其对Wnt/β-catenin信号通路转录活性的影响。方法:采用PCR克隆全长野生型HBx基因,在此基础上,分别构建融合增强型绿色荧光蛋白(EGFP)基因和缺失HBx基因C端49~154 aa、C端85~154 aa、N端1~57 aa+C端141~154 aa、N端1~57 aa或N端1~84 aa截短突变体的表达载体;激光共聚焦显微镜下观察HBx截短突变体在HEK293细胞中的定位;采用萤光素酶报告系统检测HBx截短突变体对Wnt/β-catenin信号通路转录活性的影响。结果:(1)成功构建了HBx截短突变体与EGFP基因融合的表达载体。(2)野生型HBx及截短突变体在HEK293细胞中具有不同的定位,其中N端缺失突变体主要定位于细胞核周胞质;C端缺失突变体在胞质胞核中呈均匀分布;N端+C端缺失突变体的定位类似于野生型HBx,主要定位于胞质,呈不均一的粗大颗粒,胞核中也有少量表达。(3)与野生型HBx相比,HBx C端49~154 aa、C端85~154 aa、N端1~57 aa+C端141~154 aa、N端1~57 aa和N端1~84 aa缺失突变体在Huh7细胞中均使Wnt/β-catenin信号通路的转录活性明显下降,分别下降了78.7%、84.7%、75.7%、93.8%和95.5%。结论:HBx不同截短突变体的细胞定位及对Wnt/β-catenin信号通路转录活性不同,提示HBx的不同功能区对Wnt信号的转录激活发挥着不同的作用。AIM： To observe the intracellular location of hepatitis B virus X protein （HBx） truncated mutants and their impacts on the transcriptional activity of 13-catenin/T-cell factor 4 （TCF4） in Wnt signaling pathway. METHODS： The full length of wild-type HBx gene was cloned by PCR. The C-terminal of 49-154 aa （HBxI ）, C-termi- nal of 85 - 154 aa （ HBx2 ）, N-terminal of 1 - 57 aa and C-terminal 141 - 154 aa （ HBx3 ）, N-terminal of 1 - 57 aa （ HBx4 ）, and N-terminal of 1 - 84 aa （ HBx5 ） deletion mutants of HBx gene fused with enhanced green fluorescent protein （EGFP） gene （fl-IBxl, fHBx2, fHBx3, fHBx4 and fHBxs, respectively） were constructed. The intracellnlar locations of HBx truncated mutations were observed under confocal laser scanning microscope. Moreover, the effects of HBx truncated mutants on 13-eatenin/TGF4 transcriptional activity in Wnt signaling pathway were detected by luciferase reporter system. RESULTS ： The expression vectors of HBx truncated mutants fused with EGFP gene were successfully constructed. Expres- sion of wild-type HBx and its truncated mutants in HEK293 cells had distinct dual intracellular localizations. N-terminal deletion mutants localized mainly in perinuclear cytoplasm of transfected HEK293 cells. C-terminal deletion mutants were distributed evenly both in the nucleus and cytoplasm. Similar to the cellular localization of wild-type HBx, N-terminal and C-terminal deletion mutant localized mainly in the cytoplasm in a manner of heterogeneous coarse particles, and was alsoexpressed a little in the nucleus. Compared with wild-type HBx, either N-terminal or C-terminal truncated HBx mutants in- hibited 13-catenin/TCF4 transcriptional activity, with the inhibitory rate of 78.7% （fHBxI ）, 84.7% （fI-IBx2 ）, 75.7% （fHBx3 ）, 93.8% （fHBx4 ） and 95.5% （fHBx5 ）, respectively. CONCLUSION： HBx truncated mutants show discre- pant intracellular locations and different impacts on ^-catenin/TCF transcriptional activity in Wnt signaling pathwa

关 键 词：乙型肝炎病毒X蛋白 截短突变体 细胞内定位 转录活性 

分 类 号：R373.21[医药卫生—病原生物学] R394.3[医药卫生—基础医学]