| 马云 王昌博 李斌 元秦 凌雪 付亮 董晓 何淑雅.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[J].,2014,14(28):5407-5435 |
| 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 Effectionof Ganglioside GM1 |
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| DOI： |
| 中文关键词: 脆性X相关基因1 真核表达 神经节苷脂 |
| 英文关键词: Fragile-X-related gene 1 Eukaryotic expression vector GM1 |
| 基金项目:国家自然科学基金项目(81302079)；广州医科大学青年基金项目(2012C10)；
广州医科大学附属第三医院青年基金项目(2012Y15) |
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| 中文摘要: |
| 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EcoRI和 XhoI 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。 |
| 英文摘要: |
| 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 of EcoRI and XhoI. 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 GM1 was increased (P＜0.05) considerably in SH-SY5Y cells
transfected with pcDNA3.1 (-)-FXR1.Conclusion: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 mechanismin fragile X syndrome. |
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