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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。
AIM: To construct and identify shRNA vectors targeting tubulin cofactor A (TBCA) gene. Methods: According to the human TBCA mRNA sequence, two oligonucleotides encoding the shRNA were designed and synthesized. After annealed to form the oligonucleotide template, the two fragments were cloned into pSilencer2.0-U6-neo and pGCsi-U6 -neo-GFP vector, identified by restriction enzyme digestion, and western blot was performed after liposome transfection. Results: The restriction enzyme digestion confirmed the successful construction of interference plasmids. The green fluorescent protein was observed 24 hours after transfection with 786-0 cell line. Western blot confirmed that TBCA expression decreased. Conclusion: The shRNA expression vector targeting TBCA gene was successfully constructed, which laid the foundation for the further research on RNAi.