IKKβ-RNAi腺病毒构建及其HEK293T细胞表达

CONSTRUCTION OF HUMAN IKKβ-TARGETING RNAI ADENOVIRUS VECTOR AND ITS SUPPRESSIVE EFFECT ON THE EXPRESSION OF IKKβIN HEK293T CELLS

目的构建沉默人IκB激酶β(IKKβ)基因表达的RNA干扰(RNAi)腺病毒,了解其对IKKβ基因表达的作用。方法根据GenBank IKKβ基因的mRNA,利用RNAi Designer工具软件设计合成4对IKKβ基因的特异性小干扰RNA(siRNA),合成互补的寡核苷酸链,构建重组腺病毒,鉴定目的基因。构建IKKβ基因过表达质粒,以与RNAi腺病毒质粒共同转染HEK293T细胞作为实验组,不转染任何质粒的细胞作为空白对照组,共转染过表达质粒和阴性对照病毒载体质粒的HEK293T细胞为阴性对照组,蛋白免疫印迹(Western Blot)方法检测各组HEK293T细胞中IKKβ基因表达。结果成功构建IKKβ-RNAi腺病毒质粒及IKKβ过表达质粒,实验组IKKβ基因表达较空白对照组及阴性对照组下降,差异有显著性(t=49.18、44.648,P<0.05)。结论构建的IKKβ-RNAi腺病毒质粒能有效抑制IKKβ基因表达,为进一步探讨肿瘤的生物治疗提供理论基础。

Objective To construct human IKKβ-targeting RNAi adenovirus vector and observe their gene silencing effect on IKKβ. Methods Four pairs of IKKi3 gene-specific small interfering RNA （siRNA） were designed using the RNAi De- signer software according to mRNA of IKKβ gene complementary oligonucleotides were synthesized, recombinant adenovirus con- structed and target gene identified successfully. Co-transfected HEK293T cells with RNAi adenovirus vector and Over-expression IKKβ plasmid served as the experimental group, those without any plasmid transfeeted cells as blank control group and ceils co- transfected expression plasmids and negative viral vectors as a negative control group. The expressions of IKKβ gene in HEK293T cells in each group were detected using Western Blot. Results RNAi adenovirus vector and over-expression IKKβ plasmid were successfully created, the IKKβ gene expression in the experimental group declined versus both blank-and negative-control groups, the differences being significant （t=49.18,44.648 P〈0.05）. Conclusion The constructed IKKβ-RNAi adenovirus plasmid can effectively inhibit the expression of IKKβ. This research provides a theoretical basis for a further study of cancer biotherapy.