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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 eukaryotic expression vectors of MJD1 wild type and CAG truncated truncated mutant MJD1 respectively, and to clarify whether the polyglutamine expansion mutation of atax-in-3 can lead to the formation of protein aggregates in the nucleus. Methods: The coding sequences of wild-type MJD1 and CAG trinucleotide repeat-extended mutant MJD1 were amplified by PCR using pAS2-1-M JD20Q and pAS2-1-M JD68Q as template respectively. The PCR product was double-digested with BamH I and Hind III and ligated into pcDNA3.1-Myc-His (-) B. The recombinant plasmid pcDNA3.1-Myc-H is (-) BM JD20Q was identified by restriction enzyme digestion and DNA sequencing pcDNA3.1-Myc-H is (-) BM JD68Q. The recombinant plasmids were transfected into SH-SY5Y cells and the expression of ataxin-3 was verified by Western blotting. The formation of nuclear protein aggregates in transfected cells was observed by laser confocal microscopy. Results: Enzyme digestion and DNA sequencing confirmed that the target gene was cloned into pcDNA3.1-Myc-His (-) B; its expression in transfected cells was verified by western blotting; SH-SY5Y cells transfected with pcDNA3.1 .1-Myc-His (-) BM JD68Q was found in the formation of nuclear protein aggregates, but no nuclear protein aggregates formed after transfection with pcDNA3.1-Myc-His (-) BM-JD20Q. CONCLUSION: The eukaryotic expression plasmid of MJD1 was successfully constructed. The polyglutamine extension mutation of ataxin-3 can lead to the formation of protein aggregates in the nucleus.