TNFAIP8干扰质粒的构建及最佳干扰效率质粒的筛选

Construction and identification of interference plasmid targeting on TNFAIP8

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 the high efficiency interference plasmid of TFAIP8-shRNA-pSIREN- RetroQ. Methods： Selected and synthesized three Target Sequence of TNFAIP8 shRNA1, TNFAIP8 shRNA2, TNFAIP8 shRNA3, and construct the TNFAIP8 interference plasmid. Transfection TNFAIP8-shRNA-pSIREN-RetroQ interference plasmid to A549 cells. Filter out the highest interference efficiency plasmid by detecting the mRNA and protein levels using RT-PCR and Western blot methods. Results： We successfully design and built three TNFAIPS-shRNA-pSIREN -RetroQ interference plasmids,and screen out the highest efficiency interference plasmid. Conclusion： Three interference plasmids targeting the TNFAIP8 gene have been constructed successfully and provide a useful tool for studying the function of TNFAIP8.