MJD1基因真核表达载体的构建及其在SH-SY5Y细胞中的表达

Construction of the eukaryotic expression vector of MJD1 and its expression in SH-SY5Y cells

目的:分别构建野生型MJD1以及CAG三核苷酸重复扩展突变型MJD1的真核表达载体;明确atax-in-3的多聚谷氨酰胺扩展突变是否可导致细胞核内蛋白聚合体形成。方法:分别以pAS2-1-M JD20Q和pAS2-1-M JD68Q为模板,通过PCR扩展野生型MJD1以及CAG三核苷酸重复扩展突变型MJD1的编码序列。PCR产物经BamH I和Hind III双酶切后连入pcDNA3.1-Myc-H is(-)B,通过酶切以及DNA测序鉴定重组质粒pcDNA3.1-Myc-H is(-)B-M JD20Q和pcDNA3.1-Myc-H is(-)B-M JD68Q。重组质粒转染SH-SY5Y细胞,通过W estern印迹验证ataxin-3的表达,应用激光共聚焦显微镜观察转染细胞的核内蛋白聚合体形成情况。结果:酶切和DNA测序证实目的基因已被克隆入pcDNA3.1-Myc-H is(-)B;其在转染细胞中的表达亦经W estern印迹得以验证;SH-SY5Y细胞转染pcDNA3.1-Myc-H is(-)B-M JD68Q后可见核内蛋白聚合体形成,而转染pcDNA3.1-Myc-H is(-)B-M JD20Q后未见核内蛋白聚合体形成。结论:成功构建了MJD1的真核表达质粒;ataxin-3的多聚谷氨酰胺扩展突变可导致细胞核内蛋白聚合体形成。

Objective To construct the eukaryotic expression vector of MJD1 with normal copies of CAG trinucleotide repetition and MJD1 with CAG trinucleotide repetition expansion mutation respectively, and to determine whether the polyglutamine expansion in ataxin-3 could lead to the formation of intranuclear aggregation. Methods The coding sequence of wild-type MJD1 and mutant MJD1 was amplified by PCR from pAS2-1-MJD20Q and pAS2-1-MJD68Q respectively. After being digested with BamH I and Hind III, the PCR products were inserted into pcDNA3.1-Myc-His（-）B. The recombinant plasmids pcDNA3.1-Myc-His（-）B-MJD20Q and pcDNA3. 1-Myc-His （-）B-MJD68Q were identified by enzyme digestion analysis and DNA sequencing. The recombinant plasmid was transfected into SHSYSY cells and the expression of MJD1 in the transfected cells was analyzed by Western blot. The immunofluorescence of the transfected cells was examined using a confocal microscope to observe the formation of intranuclear aggregation. Results Enzyme digestion analysis and DNA sequencing showed that the target gene was cloned into pcDNA3.1-Myc-His（-）B. The expression of MJD1 in the transfected cells was confirmed by Western blot; The SH-SYSY cells transfected with pcDNA3. 1-Myc-His （-） B-MJD68Q showed the formation of intranuclear aggregation, but the cells transfected with pcDNA3.1-Myc-His（-）B-MJD20Q did not show such phenomenon. Conclusion The eukaryotic expression vectors of MJD1 has been successfully constructed; The polyglutamine expansion in ataxin-3 could lead to the formation of intranuclear aggregation.