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目的:构建并筛选出干扰效率最佳的TNFAIP8-shRNA-p SIREN-Retro Q干扰质粒。方法:通过生物软件选择3个TNFAIP8基因干扰位点,构建干扰质粒并测序验证,将干扰质粒及对照质粒分别转染至A549细胞,通过RT-PCR、Western blot检测干扰效率。结果:经RT-PCR和Western-Blot证实TNFAIP8-shRNA-p SIREN-Retro Q干扰质粒能有效干扰并抑制细胞内TNFAIP8基因的表达,通过流式检测发现降低TNVAIP8表达可以提高细胞对a DR5Sc Fv诱导凋亡的敏感性。结论:成功构建和设计了对TNFAIP8基因具有显著干扰效率的干扰质粒,为进一步研究TNFAIP8基因的功能奠定了基础。
Objective: To construct and screen TNFAIP8-shRNA-p SIREN-Retro Q interfering plasmid with the best interference efficiency. Methods: Three TNFAIP8 gene interference sites were selected by biological software. The interference plasmids were constructed and verified by sequencing. The interference plasmids and the control plasmids were transfected into A549 cells respectively, and the interference efficiency was detected by RT-PCR and Western blot. Results: RT-PCR and Western-Blot confirmed that TNFAIP8-shRNA-p SIREN-Retro Q interference plasmid could effectively interfere with and inhibit the expression of TNFAIP8 in cells. Flow cytometry showed that reducing TNVAIP8 expression could enhance the induction of a DR5Sc Fv Apoptosis sensitivity. Conclusion: The interference plasmids with significant interference efficiency to TNFAIP8 gene were successfully constructed and designed, which laid the foundation for further study on the function of TNFAIP8 gene.