N-乙酰氨基葡萄糖转移酶干扰慢病毒载体系统的建立及应用

Construction and application of lentiviral-based system against N-Acetylglucosaminyltransferase

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 lentiviral-based RNA interference （RNAi） system against N-Acetylglucosami- nyl transferase （GnT） Ⅲ, Ⅳa and Ⅴ, and detect the inhibition of the lentiviruses on different GnT expression in mouse hepatoma cells. Methods Specific shRNA sequences were designed based on the three gene sequences and lentivira] vectors were constructed. Lentivial particles were obtained using 293T packaging cells. Then the expression of the three GnTs was detected by RT-PCR and Western blot after the infection of lentivirus. Results Sequencing results showed that the three lentiviral expression vectors were successfully constructed, and high titers of lentivirus were obtained. The expression of the three GnTs was inhibited effectively after infection of the interference lentiviral particles. Conclusion Lentiviral RNAi can effectively inhibit the expression of GriT-Ⅲ, GnT-Ⅳa and GnT-Ⅴ.