葡萄糖苷酶Ⅰ特异性siRNA-EGFP真核表达载体的构建及表达

Construction and Expression of GlucosidaseⅠSpecific siRNA Eukaryotic Expression Vector

目的:构建葡萄糖苷酶Ⅰ(glucosidaseⅠ, GluⅠ)特异性siRNA真核表达载体。方法:根据 siRNA靶序列选取原则,设计并合成针对鼠葡萄糖苷酶ⅠcDNA的寡聚DNA,退火并连接至载体 pSilencer1.0 U6,再将 U6 启动子及下游插入片断一起切下,克隆至真核表达载体 pEGFP C1,通过限制性酶切和测序对重组表达载体进行鉴定,最后将该质粒转染至HeLa细胞,共聚焦荧光显微镜观察其表达。结果:①限制性酶切和测序结果表明成功构建了带有EGFP基因的鼠GluⅠ特异性的siRNA真核表达载体 pC 1U6glu; ②共聚焦荧光显微镜观察结果表明p C1U6glu成功转染至HeLa细胞。结论:pC 1U6glu的成功构建及表达为进一步利用 siRNA研究葡萄糖苷酶Ⅰ的功能和治疗肿瘤奠定基础。

Objective: To construct eukaryotic expression vector of siRNA specific for rat glucosidaseⅠ. Methods: According to the selection guideline of siRNA target sequence, oligonucleotides were designed and synthesized, annealed and inserted into pSilencer 1.0-U6 , subsequently. The U6 promoter linked with the sequence coding glucosidaseⅠ-siRNA was subcloned into pEGFP-C1. The recombinant plasmid was confirmed by restriction enzyme digestion and DNA sequencing. The recombinant eukaryotic expression vector was transfected into HeLa cells and detected by the confocal fluorescence microscope. Results: The construction of glucosidaseⅠ-specific siRNA eukaryotic expression vector pC-1U6glu and its transfection into HeLa cells were confirmed successfully by the restriction digestion and confocal microscope analysis. Conclusion: The expression of pC-1U6glu in HeLa cells lays the foundation by using siRNA to study the function of glucosidaseⅠand its further application for tumor therapy.