人eya2基因小干扰RNA表达载体的构建及表达

Conctruction and Expression of Small Interference RNA Expression Vectors Targeting Human Gene eya2

目的:设计并构建人eya2(eyes absent2)基因小干扰RNA(siRNA)的真核表达载体,并观察其沉默效果。方法:以人eya2为靶基因,以pSliencer2.1-U6neo质粒为载体,根据人eya2的cDNA序列,设计含有小发卡结构的2条寡核苷酸序列,将其克隆到siRNA表达载体上;转化大肠杆菌DH5α菌株,抽提质粒,测序分析;将重组质粒转染人胚肾293T细胞,通过荧光分析、Western blot和转录活性实验检测其抑制效果。结果:重组体测序结果与目的序列相一致,证明构建了eya2 siRNA真核表达载体;荧光观察表明siRNA能显著减弱细胞中绿色荧光强度,抑制eya2基因表达;Western blot分析证明构建的siRNA能有效抑制外源性及内源性eya2基因表达;转录活性测定表明,构建的siRNA能有效抑制eya2基因表达。结论:构建了eya2 siRNA真核表达载体,该siRNA能有效地抑制eya2基因表达。

Objective： To design and construct the eukaryotic expression vector of human eyes absent 2 （eya2） small interfering RNA（siRNA） and to transfect 293T cells, investigate the silencing effect of eya2 siRNA on the expression of eya2 gene. Methods： siRNAs were designed and constructed by targeting eya2 gene using pSliencer 2.1-U6 neo based on human eya2 cDNA sequence. Two pairs of oligonucleotides were synthesized and inserted into pSliencer 2.1-U6 neo to generate siRNA eukaryotic expression vectors. E.coli DH5a strains were transformed, plasmids were extracted, and the recombinant sequences were identified. Then human 293T cells were cotransfected with the recombination plasmids and FLAG-tagged eya2 expression vector. The dumbness effect of eya2 siRNAs on the expression of eya2 gene was identified by fluorescence intensity, Western blot and analysis of transcriptional actvity. Results： The expression vectors of eya2 gene siRNAs were constructed and confirmed by DNA sequencing, eya2 siRNAs could effectively inhibit the exogenous and endogenous expression of eya2 gene by Western blot. Fluorescence microscope showed that siRNAs could inhibit the eya2 expression and obviously weaken the fluorescence intensity in the 293T cells. Conclusion： The eukaryotic expression vectors of eya2 siRNAs were constructed successfully. The siRNAs could effectively inhibit the expression of eya2 gene.