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目的构建并鉴定靶向果蝇同源蛋白3(tribbles-related protein 3,Trb3)基因的短发夹状RNA(short hairpin RNA,shRNA)真核表达载体,并检测其对靶基因的沉默效应。方法根据Genbank数据库中Trb3序列,设计、合成3对互补并特异性编码其shRNA序列的寡核苷酸,克隆到线性化的pSUPER.retro.puro质粒载体中,并对重组质粒进行酶切分析及测序鉴定;脂质体介导重组质粒和pSUPER.retro.puro Vector转染舌鳞状细胞癌Tca8113细胞株,RT-PCR和Western-Blot检测其对靶基因Trb3的表达影响。结果酶切及测序鉴定重组质粒pSUPER.puro-shTrb3序列正确;转染重组质粒后舌鳞状细胞癌Tca8113细胞中Trb3 mRNA和蛋白表达水平相对于空载体组明显下降,shRNA可以特异性沉默Trb3基因。结论成功构建了靶向人Trb3基因的shRNA真核表达载体,为进一步研究Trb3在舌鳞状细胞癌和其他头颈肿瘤中的作用奠定了基础。
Objective To construct and identify a short hairpin RNA (shRNA) eukaryotic expression vector targeting to the gene TRF3 in Drosophila melanogaster and to detect its silencing effects on target genes. Methods According to the sequence of Trb3 in Genbank, three pairs of oligonucleotides were designed and synthesized, and their shRNA sequences were designed and cloned into the linearized pSUPER.retro.puro plasmid vector. The recombinant plasmid was digested with restriction endonuclease The expression of Trb3 in the tongue squamous cell carcinoma cell line Tca8113 was detected by RT-PCR and Western-Blot. Results The sequence of recombinant plasmid pSUPER.puro-shTrb3 was confirmed by restriction enzyme digestion and sequencing. The expression of Trb3 mRNA and protein in Tca8113 cells was significantly decreased after transfected with the recombinant plasmids. ShRNA can specifically silence the Trb3 gene . Conclusion The shRNA eukaryotic expression vector targeting human Trb3 gene was successfully constructed, which laid the foundation for the further study on the role of Trb3 in tongue squamous cell carcinoma and other head and neck cancers.