野生型与突变型PS1真核表达载体的构建及其在SH-SY5Y细胞中的表达

目的为了对我们发现的中国人家族性阿尔茨海默病早老素-1(presenilin1,PS1)基因新的点突变进行蛋白功能研究,分别构建野生型和突变型PS1(G289T)与绿色荧光蛋白(EGFP)共表达载体,并检测其在SHSY5Y细胞内的表达。方法利用含人全长PS1cDNA的pcDNA3·1(zeo ),采用定点突变技术,构建PS1(G289T)-pcDNA3·1(zeo )载体。采用基因重组技术构建野生型和突变型PS1与绿色荧光蛋白共表达载体。应用脂质体将携带野生型和突变型PS1的质粒转染至SH-SY5Y细胞,检测报告基因表达,RT-PCR检测PS1mRNA表达。结果经限制性内切酶酶切图谱分析及DNA测序证实融合蛋白表达载体构建成功;RT-PCR产物经测序显示突变型PS1mRNA在SH-SY5Y中有表达。结论成功构建了人野生型和突变型PS1与EGFP共表达载体并成功转染至SH-SY5Y细胞,为进一步的研究工作奠定了基础。

Construction of eukaryotic expression vector containing PS1 and the expression in neuroblastoma cell line SH-SY5Y

Objective To study the function of a new missense mutation in the PS1 gene carried by a Chinese pedigree, we construct an eukaryotic expression vectorcontaining wild or mutation type PS1-EGFP, and established a stable SH-SY5Y cell line expressing the fusion protein. Methods Mutant human PS1(PS1 V97L) cDNA was generated based on pcDNA3.1(zeo ) plasmid containing total length of human wild type PS1 using site-directed mutagenesis method. The EGFP sequence was subcloned into the wild or mutant pcDNA3.1/PS1 vector to construct co-expressing vectors. Vectors construct containing the cDNA encoding either for human PS1 wt or PS1 V97L mutation and pcDNA void vector were introduced into SH-SY5Y cells using LipofectamineTM methodology. Expression of the report gene EGFP was examined under fluorescence microscope. The expression of PS1 in nontransfected(NT) and transfected cell lines was checked by RT-PCR. Results Enzyme digestion analysis and DNA sequencing showed that the fusion protein expressing vectors were constructed successfully. The mutant PS1 mRNA expression was identified and confirmed by RT-PCR. Conclusion The vectors containing human wild or mutant PS1 and EGFP were successfully constructed and transfected into SH-SY5Y cells. This study provides a useful cell model to further study.