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目的构建针对N-乙酰氨基葡萄糖转移酶(GnT)Ⅲ、Ⅳa和Ⅴ的RNA干扰(RNAi)慢病毒系统,并检测干扰慢病毒在体外小鼠肝癌细胞中对不同GnT表达的抑制作用。方法针对三种基因序列设计合成特异的shRNA序列,并构建干扰慢病毒表达载体,利用病毒包装细胞293T包装生产病毒,感染靶细胞Hca-F后,应用RT-PCR和免疫印迹检测干扰慢病毒对三种N-乙酰氨基葡萄糖转移酶表达的抑制。结果经测序证实三种干扰慢病毒表达载体构建成功,并获得高滴度的感染慢病毒。干扰慢病毒感染靶细胞后能够有效下调三种N-乙酰氨基葡萄糖转移酶的表达。结论干扰慢病毒可有效地抑制三种N-乙酰氨基葡萄糖转移酶GnT-Ⅲ、GnT-Ⅳa和GnT-Ⅴ的表达。
Objective To construct a RNA interference (RNAi) lentiviral system targeting N-acetylglucosaminyltransferase (GnT) Ⅲ, Ⅳa and Ⅴ and to detect the inhibitory effect of lentivirus on the expression of different GnT in mouse hepatocellular carcinoma cells in vitro. Methods The specific shRNA sequence was designed and synthesized based on the three gene sequences. The recombinant lentiviral vector was constructed and transfected into 293T cells by virus packaging cells. After infected with Hca-F, RT-PCR and Western blotting were used to detect lentivirus Inhibition of expression of three N-acetylglucosaminyltransferases. Results Sequencing confirmed that the three lentiviral vectors were constructed successfully and got high titer of lentivirus. Interfering lentivirus-infected target cells can effectively down-regulate the expression of three N-acetylglucosaminyltransferases. Conclusion Interfering lentivirus can effectively inhibit the expression of GnT-Ⅲ, GnT-Ⅳa and GnT-Ⅴ in three N-acetylglucosamine transferases.