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目的: 确立基于Gal4/ vp16- UAS 和双荧光素酶报告基因系统检测??- 分泌酶切割淀粉样前体蛋白活性的方法。方法: 将插入上游激活序列(UAS) 和萤火虫荧光素酶报告基因的质粒MH100, 嵌合酵母活性转录因子( Gal4) 、单纯疱疹病毒蛋白( VP16) 和??- 分泌酶切割位点的质粒C99- GVP, 以及海肾荧光素酶质粒pRL- CMV, 用脂质体转染法转入稳定表达淀粉样前体蛋白C 末端的人神经母细胞瘤细胞( SH- SY5Y) , 用免疫沉淀Western blot 分析法检测??- 淀粉样蛋白( A??) 的生成, 利用Gal4/ vp16- UAS 和双荧光素酶报告基因系统测定荧光素酶报告基因的表达。结果: 免疫沉淀Western blot 分析表明A( 的生成在??- 分泌酶激活剂神经节苷脂GM1 作用下升高并呈剂量依赖性, 同时双荧光素酶法检测??- 分泌酶活性也同步升高。在??- 分泌酶抑制剂作用下 A?? 的产生呈剂量依赖性的减少, 同时??- 分泌酶活性也同步降低。结论: 基于Gal4/ vp16- UAS 和双荧光素酶报告基因系统检测 ??- 分泌酶活性的方法有效可靠, 是一种敏感、定量的检测方法。

英文摘要:

Objective: To establish an assay, based on Gal4/ vp16- UAS and Dual- Luciferase reporter gene, for detecting the activity of ??- secretase in the proteolytic processing of precursor protein. Methods: Human neuroblastoma cells SH - SY5Y C99, which stably expresses APP C99, were transfected with the plasmidsMH100, C99- GVP and pRL- CMV. MH100 encodes the up activated sequence ( UAS) and luciferase. C99- GVP encodes the C- terminal fragment of APP and Gal4/ VP16. pRL- CMV encodes the Renilla luciferase. Immunoprecipitation Western blot was used to analyze the A?? secretion. The expression of the reporter gene in the cell lysates was assayed by the method based on Gal4/ VP16- UAS and Dual- Luciferase reporter gene system. Results: Luciferase activities detected by the Gal4/ VP16- UAS and Dual- Luciferase reporter gene assay system were increased by GM1, in parallel with the increase of A?? secretion detected by immunoprecipitation Western blot. ??- Secretase Inhibitor IX inhibited the secretion of A?? and it also decreased luciferase activities in a dose dependent manner. Conclusion: The assay based on Gal4/ VP16- UAS and Dual- Luciferase reporter gene is effective and reliable for detecting the activity of ??- secretase with sensitivity and quantitation, which is helpful to the researches of pathomechanism and therapy of Alzheimer?? s disease.

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