Blimp1基因突变影响其编码蛋白转录抑制功能的体外研究

Influence of Blimp1 mutation on its transcriptional repressor activity: An in vitro study

目的:探讨Blimp1基因突变在弥漫大B细胞淋巴瘤(diffuse large B-cell lymphoma,DLBCL)发病中的作用。方法:应用基因测序方法检测1例DLBCL患者Blimp1基因的突变情况;用PCR扩增其Blimp1全长编码基因,并通过定点突变获得野生型Blimp1的全长编码基因。分别将野生型、突变型Blimp1的全长编码基因亚克隆至含有Flag标签的Flag-CMV4真核表达载体中,将重组质粒分别命名为Flag-Blimp1-wt和Flag-Blimp1-mut;采用PCR扩增获得人CIITA启动子,并将扩增片段插入PGL3-Basic荧光报告基因载体中,所有重组质粒均通过酶切鉴定并测序验证序列正确;将Flag-Blimp1-wt/mut分别与PGL3-CIITA-promoter共转染293T细胞,用荧光素酶报告基因系统检测报告基因载体的活性及Blimp1基因对其表达的调控,采用蛋白印迹法检测融合蛋白Flag-Blimp1-wt与Flag-Blimp1-mut的蛋白表达差异。结果:成功构建野生型、突变型Flag-Blimp1融合蛋白表达载体及PGL3-CIITA-promoter报告基因载体;经荧光素酶报告基因检测系统证实,构建的报告基因载体具有启动子活性,野生型Blimp1蛋白对其表达具有抑制作用,且该抑制作用与Blimp1的转染剂量呈正相关,而突变型Blimp1对其抑制功能明显减弱;蛋白印迹检测结果显示,突变型Blimp1蛋白明显低于野生型。结论:该例患者中Blimp1的突变影响了其转录抑制功能,可能是由基因突变导致Blimp1蛋白表达量的减少所引起。

Objective To study the role of Blimpl mutation in the development of diffuse large B cell lymphoma （DLBCL）. Methods Mutation of Blimpl in a DLBCL patient was detected by geue sequence analysis. Coding sequence（CDS） of Blimpl was amplified by PCR and the CDS of wide-type Blimpl was acquired using Site-directed Gene Mutagenesis Kit. The wide-type and mutant Blimpl were then ligated into Flag-CMV4, respectively, to form the recombinant plasmid Flag- Blimpl-wt and FlagBlimpl-mut;CIITA promoter was amplified by PCR and then ligated into luciferase reporter gene PGL3- Basic Vector to get PGL3-C Ⅱ TA-promoter. All the recombinant plasmids were identified by digestion and sequencing; Flag- Blimp 1-wt/mut and PGL3-C Ⅱ TA-promoter were co-transfected into 293T cells using PolyFect, and the luciferase activity was detected by using ONE-Glo^TM Luciferase Assay System, the level of Flag-Blimpl-wt/mut protein was detected by Western blotting. Results The plasmids Flag-Blimpl-wt/mut and PGL3-C Ⅱ TA-promoter were successfully constructed. The luciferase reporter system verified that PGL3-CⅡ TA-promoter plasmid had the promoter activity and the activity could be repressed by Flag-Blimpl-wt in a dose-dependent manner. However, the repressor function of Flag-Blimpl-mut was dramatically reduced. The results of Western blotting showed that the level of protein Flag-Blimpl-mut was apparently lower than Flag-Blimpl-wt. Conclusions Mutation of Blimpl impairs its transcriptional repressor activity, which may be due to the low protein level resulting from the mutation, this finding may shed a new light on further studies on the pathogenesis of DLBCL.