FXR1真核表达载体的构建与表达及其对神经节苷脂浓度的影响  

Construction and Expression of Eukaryotic Vector of FXR1 and the Effection of Ganglioside GM1

作　　者：马云[1] 王昌博[1] 李斌元[1] 秦凌雪[1] 付亮[1] 董晓[1] 何淑雅[1] 

机构地区：[1]南华大学生物化学与分子生物学研究所,湖南衡阳421001

出　　处：《现代生物医学进展》2014年第28期5407-5409,5435,共4页Progress in Modern Biomedicine

基　　金：国家自然科学基金青年项目(81200881);湖南省自然科学基金项目(12JJ6073)

摘　　要：目的:构建脆性X相关基因1(FXR1)的真核表达载体并检测其对神经节苷脂(GM1)浓度的影响。方法:以pYESTrp3-FXR1为模板,利用PCR扩增FXR1基因,PCR产物经EcoR I和Xho I双酶切后插入真核表达载体pcDNA3.1(-)中,获得的阳性克隆进一步酶切及测序鉴定;将构建成功的pcDNA3.1(-)-FXR1转染SH-SY5Y细胞后,采用Western blot检测FXR1的表达情况,同时采用ELISA试剂盒检测细胞内GM1的浓度。结果:PCR扩增产物为1.9 Kb的片段,与FXR1基因大小相符,阳性克隆经双酶切后获得两条分别为5.4 Kb和1.9 Kb的片段,测序结果与GeneBank中序列相同。构建成功的重组质粒pcDNA3.1(-)-FXR1转染SH-SY5Y细胞后,细胞中FXR1的表达增加,同时有效提高了细胞内GM1的浓度(P<0.05)。结论:成功构建了真核表达载体pcDNA3.1(-)-FXR1,FXR1的表达增加可以提高SH-SY5Y细胞中的GM1浓度,这些为后续深入研究FXR1基因在神经组织发育中的调控功能及其在脆性X综合征(FXS)中的作用机制奠定了基础。Objective： To construct a eukaryotic expression vector encoding FXR1 and detect its effect on the concentration of GM1. Methods： FXR1 was amplified by PCR using pYESTrp3-FXR1 as template and then inserted into eukaryotic expression vector pcDNA3.1 （-） using restriction site ofEcoR I and Xho I. The positive clones were further identified by enzyme cleaving and sequencing. The pcDNA3.1 （-）-FXR1 was transfected into SH-SY5Y cells and the expression level of FXR1 was detected by Western blotting and the concentration of GM1 was detected by ELISA Kit. Results： The PCR product was a 1.9 Kb fragment consistent with FXR1 gene size, the positive clones were double enzyme cleaved to 5.4 Kb and 1.9 Kb fragment and the results of sequencing were the same as GeneBanks. The expression level of FXR1 was up-regulated and the concentration of GM 1 was increased （P 〈 0.05） considerably in SH-SY5Y cells transfected with pcDNA3.1 （-）-FXR1. Conclusions： Eukaryotic expression vector encoding FXR1 was successfully constructed, and over-expression of FXR1 could regulate the GM1 concentration, which laid the foundation for the further studying the regulatory functions of FMR1 gene in neural tissue development and the mechanism in fragile X syndrome.

关 键 词：脆性X相关基因1 真核表达 神经节苷脂 

分 类 号：R394[医药卫生—医学遗传学]