人微管蛋白辅助因子A基因shRNA表达质粒的构建及鉴定

Establishment and identification of shRNA expressing plasmid targeting tubulin cofactor A gene

目的:构建针对微管蛋白辅助因子A(TBCA)基因的shRNA载体并进行鉴定。方法:针对人TBCA基因mRNA序列,设计合成编码shRNA的两条寡核苷酸链,经退火形成发卡寡核苷酸模板片段,经双酶切克隆至pSilencer2.0-U6-neo和pGCsi-U6-neo-GFP载体,进行酶切测序鉴定,脂质体转染后进行western blot测定。结果:酶切测序证实成功构建干扰质粒,脂质体转染786-0细胞系后24小时可见绿色荧光蛋白。Western blot证实TBCA表达量降低。结论:成功构建了针对TBCA基因shRNA表达载体,为下一步进行RNAi的相关研究奠定了基础。

Objective：To construct the expressing vector encoding small hairpin RNA （shRNA） targeting tu- bulin cofactor A （TBCA） gene and to identify it. Method.. According to the mRNA sequence of TBCA gene, two expression sequences of shRNA were designed and inserted into expression vector pSilencer2.0-U6-neo and pGCsi- U6-neo-GFP. The recombined plasmid vectors were identified by restriction enzyme analysis and sequencing. Af- ter transfecting into 786-0 cell, western blot was used to identify the expression of TBCA. Result： The interferen- tial plasmid was constructed correctly, which was confirmed by restriction endonuclease digestion and sequencing analysis. After transfecting 786-0 cell lines, green fluorescent protein was found. Western blot indicated that the expression of TBCA decreased. Conclusion.. The interferential plasmid was constructed successfully so as to lay a foundation for the study of RNAi.