摘要
目的 将鼠dishevelled 1(DVL 1)cDNA重组质粒转染到培养的鼠成神经瘤细胞N2a ,建立瞬时表达系统 ,为研究Wnt信号途径在阿尔茨海默病 (AD)发病中的作用提供实验基础。方法 用常规分子生物学方法扩增、纯化DVL 1cDNA重组质粒 ,用紫外分光技术检测纯化质粒的纯度 ;用脂质体介导法将重组质粒转染入培养的鼠成神经瘤细胞N2a,用免疫印迹、免疫荧光法从蛋白质水平检测转染和表达效果。结果 质粒酶切电泳结果显示 :鼠DVL 1cD NA重组质粒PCS2 +MT MDVL1扩增和纯化成功 ,纯度达到要求 ;免疫印迹、免疫荧光结果显示外源鼠DVL 1cDNA重组质粒在鼠成神经瘤细胞N2a中获得表达 ,基因转染和表达率为 5 7 6 %。结论 成功将鼠DVL 1cDNA重组质粒转染到培养的鼠成神经瘤细胞N2a中 ,并获得瞬时表达。
Objective To establish a transient expression system of mouse DVL-1cDNA recombinant plasmid in cultured wild-type mouse Neuroblastoma 2a (N2a), for the further use in studying the role of Wnt signaling in Alzheimer disease pathogenesis. Methods After being amplified and purified, the recombinant plasmid was transferred into cultured N2a by lipofectamine. The transfection and expression were examined by Western blot and immunofluorescence microscopy. Results ClaⅠdigestion revealed that recombinant plasmid PCS2+MT-MDVL1 was successfully amplified and purified, and the successful transfection and expression of the fusion protein in N2a cells was determined by using anti-c-myc tag antibody. The transfection efficiency was 57.6 % evaluated by immunofluorescence. Conclusion A transient expression system of mouse DVL-1 fusion protein was established, which can serve as a tool in studying the role of Wnt signaling in AD.
出处
《华中科技大学学报（医学版）》
CAS
CSCD
北大核心
2004年第4期398-401,共4页
Acta Medicinae Universitatis Scientiae et Technologiae Huazhong
基金
国家自然科学基金资助项目(No.39925012)
国家杰出青年科学基金资助项目(No.30100057)
国家"973"研究基金资助项目(No.G1999054007)
