人工转录因子促进外源基因在CHO细胞中的高效表达

Enhancement of Exogenous Gene Expression by Artificial Transcription Factor in CHO Cells

摘要:

以酵母转录因子GAL4中1-147位氨基酸序列为构建人工转录因子的DNA结合结构域，单纯疱疹病毒转录激活子VP16中12肽（DALDDFDLDMLG）的4个串联重复作为人工转录因子的功能结构域，用SV40的核定位序列（NLS）将两部分连接起来，构建了人工转录因子GVP4并将其克隆进入表达载体pcDNA3.1/Hygro(+) 中。将不同长度的人工转录因子结合序列构建在外源基因表达载体pcDNA3.1(+) 启动子CMV的上游，分别连接外源基因EGFP和tPA。用表达人工转录因子和外源基因的载体共转染CHO细胞，EGFP和tPA在含不同数量人工转录因子结合位点表达载体pcDNA3.1(+)转染的CHO细胞中的表达水平呈现不同程度的提高。其中，以引入10个人工转录因子结合位点的表达载体的效果最明显，EGFP和t-PA的表达效率均提高2～3倍。结果表明，人工转录因子能有效地促进外源基因在哺乳动物细胞中的表达。

Abstract:

Using the amino acids 1-147 of the yeast transcriptional activator GAL4 as the DNA_binding domain and four tandem repeats of the 12_aa peptide (DALDDFDLDMLG)of the herpesvirus as the activation domain,an artificial transcription factor,GVP4,was constructed via the linkage of the nuclear localization signal sequence of SV40.And then,GVP4 was cloned into expression vector pcDNA3.1/Hygro (+).Various amounts of targeting sites of artificial transcription factor were linked to the upstream of promoter CMV in exogenous gene expression vector pcDNA3.1(+) that separately harbored EGFP cDNA and t_PA cDNA.The CHO cells were then co_transfected with GVP4 expression vector and EGFP or t_PA expression vector.The effect of GVP4 on exogenous gene expression was evaluated by measuring the fluorescence intensity of EGFP in CHO cells and the concentration of t_PA in the supernatant.GVP4 showed positive effect on the enhancement of exogenous gene expression in CHO cells integrated with targeting sites of artificial transcription factor.And,CHO cells integrated with 10 targeting sites of GVP4 was more favorable to foreign gene expression,which resulted in 2～3_fold increase in both EGFP and t_PA expressions.These results indicated that artificial transcription factor is potent in the enhancement of exogenous gene expression in mammalian cells.