基因表达谱芯片技术筛选β干扰素基因转染细胞的反式调节基因
Screening and identification of genes trans-regulated by β-interferon protein with microarray assay
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摘要: 目的: 筛选能被β干扰素反式调节的靶基因,研究β干扰素的生物学功能及其机制。方法: 设计并合成β干扰素(IFNβ)特异性引物,应用聚合酶链反应(PCR)技术扩增IFNβ基因片段,以常规的分子生物学技术将获得的IFNβ编码基因片段克隆到TA载体中,进行测序鉴定后构建真核表达载体pcDNA3.1(-)-IFN-β。以空载体pcDNA3.1(-)为平行对照,转染HepG2细胞,制备转染后的细胞裂解液,提取mRNA。应用基因表达谱芯片技术对差异表达的mRNA进行检测和分析。结果: HepG2细胞经转染IFN-β之后,有70条差异基因表达,其中40条基因表达增强,30条基因表达降低。这些差异表达基因与细胞的增生、分化和细胞的信号转导密切相关。结论: 应用基因表达谱芯片技术筛选到IFN-β的反式调节基因,为进一步探索IFN-β可能的调节机制及其生物学功能提供了新的依据。Abstract: Objective: To screen genes regulated by IFN-βusing cDNA microarray technique. Methods: The IFN-βcoding DNA fragment was amplified with polymerase chain reaction（PCR） technique.The expressive vector of pcDNA3.1-IFN-βwas constructed by routine molecular biological methods.The HepG2 cells were transfected by pcDNA3.1（-） and pcDNA3.1-IFN-β respectively,using FuGENE6 transfection Reagent.The total RNA was isolated and reverse transcribed.The cDNAs were subjected for microarray screening with 1,152 cDNA probes. Results: The expressive vector has been constructed and confirmed by restriction enzyme digestion and DNA sequencing analysis.High quality mRNA and cDNA had been prepared and successful microarray screening had been conducted.From the scanning results,it was found 40 genes were up-regulated and 30 genes were down-regulated by IFN-β gene. Conclusion: IFN-βgene is a trans-regulator.
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