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

作　　者：刘文明[1,2] 杨晶晶 胡如意[1,2] 邱兴烽[1,2] 石春燕[1,2] 齐忠权 刘忠臣[1,2] 庄国洪 

机构地区：[1]厦门大学附属中山医院 [2]厦门大学医学院抗癌研究中心,厦门361005

出　　处：《中国免疫学杂志》2015年第5期650-654,共5页Chinese Journal of Immunology

基　　金：国家自然科学基金项目(81272720);福建省卫计委医学创新课题(2014-CXB-43);厦门市科技计划项目(3502Z2083008)资助

摘　　要：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.

关 键 词：TNFAIP8 RNA干扰 重组质粒 筛选 

分 类 号：R735.9[医药卫生—肿瘤]