shRNA慢病毒抑制BGC823细胞株GnT-V表达的载体构建

Inhibition of GnT-V gene expression in BGC823 cell line by using Lentivirus-mediated shRNA

关键词:  N-乙酰氨基葡萄糖转移酶V  RNA干扰  慢病毒

Key words:  beta-1  6-N-acetylgluco-saminyltransferase  RNA interference  Lentivirus

摘要:

目的构建针对N-乙酰氨基葡萄糖转移酶V（GnT-V）基因的shRNA（short harpin RNA,siRNA前体）表达载体,鉴定GnT-V基因干扰效率。方法体外设计并合成针对GnT-V基因的慢病毒shRNA干扰载体,通过PCR及测序证实载体构建成功。将慢病毒颗粒以最适滴度转染人低分化胃癌细胞株BGC823,应用Real-Time PCR、Western blotting检测GnT-V抑制效率。结果构建的慢病毒载体的序列通过PCR、测序等鉴定证实与设计序列相同;Real-Time PCR检测显示干扰组BGC823细胞GnT-V mRNA表达率下降88.04%,Western blotting显示干扰组BGC823细胞表达GnT-V蛋白量较空白载体对照组明显减少。结论构建的shRNA表达载体可在mRNA和蛋白水平上有效抑制BGC823细胞株GnT-V基因的表达。

Abstract:

Objective To construct the shRNA expression vector which targeting human beta-1,6-N-acetylgluco -saminyltransferase（GnT-V） gene and to detect its silencing effects in human gastric cancer cell line BGC823.Methods Three pairs of target segments were synthesized and cloned into Lentivirus-mediated shRNA respectively and the vectors were identified by enzyme digestion analysis and DNA sequencing.Then the expression vectors were transfected into BGC823 cell line.The expressions of GnT-V gene at mRNA and protein levels were detected by Real-Time PCR and Western blotting.Results Enzyme digestion analysis and DNA sequencing showed that the target segments were cloned into pLVTHM lentiviral vector respectively.Real-Time PCR detected that the expression rate of GnT-V gene of BGC823 cells in RNA interference group had dropped 88.04%as compared with that of the control.Western blotting also showed that expression of GnT-V protein was decreased in RNA interference group.Conclusion The shRNA expression vector effectively inhibits the expression of GnT-V gene at mRNA and protein levels in BGC823 cell line.