为了研究不同方向的嵌合体内含子对重组神经生长因子(Nerve growth factor,NGF)基因表达的影响,以人β-珠蛋白第一内含子5’端剪接序列和人免疫球蛋白重链可变区内含子3’端剪接序列组合而成的嵌合体内含子作为研究对象,在NGF基因5’端插入不同方向的嵌合体内含子,构建含不同方向内含子的NGF基因表达载体。转染至CHO细胞后,G418筛选稳定转染的细胞,荧光定量PCR、ELISA和Western blotting检测不同载体NGF基因的表达情况。结果显示内含子可以大幅度提高NGF基因的表达,且正向内含子对NGF基因表达的增强作用无论是在mRNA水平还是在蛋白水平都要高于反向内含子。所以内含子能够提高外源NGF基因的表达,且内含子调控转基因表达具有方向性。
The aim of this study is to investigate the effect of the chimeric intron in different directions on the expression of the nerve growth factor(NGF)in recombinant Chinese hamster ovary(CHO)cells.The chimeric intron that contained the splice sequence of the first intron of the humanβ-globin and the human immunoglobulin heavy chain variable region intron was used.NGF gene was cloned into the expression vectors containing the chimeric intron in the forward or reverse direction,followed by transfecting into CHO cells,and screened under G418 to produce the stable transfected CHO cells.Fluorescence quantitative PCR,ELISA,and Western blotting were performed to detect the recombinant NGF gene expression in CHO cells.The results showed that the chimeric introns could significantly enhance the expression of NGF in recombinant CHO cells.Moreover,the enhancing effect on NGF expression level by the intron in the forward direction showed stronger than that of the reverse direction both at mRNA and protein level.In conclusion,the chimeric intron could increase NGF expression in stably transfected CHO cells and the effect is associated with the direction of the intron insertion.
Chinese Journal of Biotechnology
Science and Technology Research Projects of Henan Province(No.182102311184)
National Natural Science Foundation of China(No.81673337).
Chinese hamster ovary cells
nerve growth factor