22R序列loop和泡突变对GFP报告基因表达的作用

Effect of 22R loop and vesical base mutation on GFP gene expression

目的:我们研究室的前期工作在猴空泡病毒40(SV40)早期mRNA PolyA序列(SV40PolyA)中发现1个活化基因的原件22R(5′-GTGAAAAAAATGCTTTATTTGT-3′),该片段可以解除Alu串连序列(或Line-1重复序列)对GFP报告基因的抑制作用。我们研究22R突变对GFP报告基因表达的作用。方法:将22R及其突变片段插入pEGFP-C1质粒,构建表达载体,瞬时转染HeLa细胞,荧光显微镜下计数观察GFP荧光阳性细胞数。结果:AAG、AGA、TGT、TTG和TGC(22R,野生型)5个质粒促进基因表达作用比较明显。VECT、VEGT、VETA、VETC、VETG和VETT明显促进GFP基因表达。结论:22Rloop和泡的突变对其活化GFP报告基因作用有重要影响。

Objective： Our work previously reported one activating gene sequences in early mRNA SV40PolyA,namely 22R（5′-GTGAAAAAAATGCTTTATTTGT-3′）which can relieve the inhibition of GFP gene induced by Alu tandem sequences.In this study,we mutated 22R to study the role of DNA structure in its activating GFP gene expression.Methods： The 22R and its mutated fragments were inserted into downstream of GFP gene in pAlu14 to construct expression vector,transfected with HeLa cells and the expression of green fluorescence protein was observed.Results： The changing loop of 22R,AAG、AGA、TGT、TTG and TGC（22R,wild type）had the effects on significantly activating GFP gene expression（marked in black bold）;changing vesical,VECT,VEGT,VETA,VETC,VETG and VETT could obviously activate GFP gene expression.Conclusion： The loop and vesical of 22R are important for 22R activating GFP gene.