不同方向内含子对重组CHO细胞中神经生长因子表达的影响（英文）
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摘要
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 human β-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.
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 human β-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.
引文
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[16]Liu XL,Zhang W,Tang SJ.Intracranial transplantation of human adipose-derived stem cells promotes the expression of neurotrophic factors and nerve repair in rats of cerebral ischemia-reperfusion injury.Int J Clin Exp Pathol,2014,7(1):174-183.
[17]Li CJ,Ma YH,Yi KL,et al.The interactions between nerve growth factor and gonadotrophins in bovine oviduct.Animal Reprod Sci,2014,149(3/4):117-123.
[18]Recillas-Targa F,Pikaart MJ,Burgess-Beusse B,et al.Position-effect protection and enhancer blocking by the chickenβ-globin insulator are separable activities.Proc Natl Acad Sci USA,2002,99(10):6883-6888.
[19]Fath S,Bauer AP,Liss M,et al.Multiparameter RNAand codon optimization:a standardized tool to assess and enhance autologous mammalian gene expression.PLoS ONE,2011,6(3):e17596.
[20]Lin Y,Li ZX,Wang TY,et al.MAR characteristic motifs mediate episomal vector in CHO cells.Gene,2015,559(2):137-143.
[21]Wang F,Zhang JH,Wang TY,et al.Regulating effects of insertion direction of matrix attachment regions on transgenic expression in stably transformed Chinese hamster ovary cells.Genet Mol Res,2015,14(2):7031-7038.
[22]Xu L,Li YH,Shi XC,et al.Expression,purification,and characterization of recombinant mouse nerve growth factor in Chinese hamster ovary cells.Protein Expr Purif,2014,104:41-49.
[23]Wang XY,Zhang JH,Sun QL,et al.Characteristic element of matrix attachment region mediates vector attachment and enhances nerve growth factor expression in Chinese hamster ovary cells.Genet Mol Res,2015,14(3):9191-9199.
[2]Fath S,Bauer AP,Liss M,et al.Multiparameter RNAand codon optimization:a standardized tool to assess and enhance autologous mammalian gene expression.PLoS ONE,2011,6(3):e17596.
[3]Williams S,Mustoe T,Mulcahy T,et al.CpG-island fragments from the HNRPA2B1/CBX3 genomic locus reduce silencing and enhance transgene expression from the hCMV promoter/enhancer in mammalian cells.BMCBiotechnol,2005,5:17.
[4]Mariati,Ng YK,Chao SH,et al.Evaluating regulatory elements of human cytomegalovirus major immediate early gene for enhancing transgene expression levels in CHO K1 and HEK293 cells.J Biotechnol,2010,147(3/4):160-163.
[5]Iwai R,Kumagai Y,Fujiwara M,et al.Combination of cytomegalovirus enhancer with human cellular promoters for gene-induced chondrogenesis of human bone marrow mesenchymal stem cells.J Biosci Bioeng,2010,110(5):593-596.
[6]Mays LE,Wilson JM.The complex and evolving story of T cell activation to AAV vector-encoded transgene products.Mol Ther,2011,19(1):16-27.
[7]Wang TY,Zhang JH,Jing CQ,et al.Positional effects of the matrix attachment region on transgene expression in stably transfected CHO cells.Cell Biol Int,2010,34(2):141-145.
[8]Wang F,Wang TY,Tang YY,et al.Different matrix attachment regions flanking a transgene effectively enhance gene expression in stably transfected Chinese hamster ovary cells.Gene,2012,500(1):59-62.
[9]Luan W.Introns and their role in gene expression.Yunnan Agr Sci&Technol,2008,(S2):182-186(in Chinese).栾薇.内含子及其在基因表达中的作用.云南农业科技,2008,(S2):182-186.
[10]Cooper AR,Lill GR,Gschweng EH,et al.Rescue of splicing-mediated intron loss maximizes expression in lentiviral vectors containing the human ubiquitin Cpromoter.Nucleic Acids Res,2015,43(1):682-690.
[11]Hermening S,Kügler S,B?hr M,et al.Increased protein expression from adenoviral shuttle plasmids and vectors by insertion of a small chimeric intron sequence.J Virol Methods,2004,122(1):73-77.
[12]Hasannia S,Lotfi AS,Mahboudi F,et al.Elevated expression of human alpha-1 antitrypsin mediated by yeast intron in Pichia pastoris.Biotechnol Lett,2006,28(19):1545-1550.
[13]Kim SY,Lee JH,Shin HS,et al.The human elongation factor 1 alpha(EF-1α)first intron highly enhances expression of foreign genes from the murine cytomegalovirus promoter.J Biotechnol,2002,93(2):183-187.
[14]Choi T,Huang M,Gorman C,et al.A generic intron increases gene expression in transgenic mice.Mol Cell Biol,1991,11(6):3070-3074.
[15]Gong YP,Tao J,Wang H,et al.Effect of chimeric intron on the Expression of Anti-bFGF antibody genes in 293Tcells.China Biotechnol,2010,30(3):9-14(in Chinese).龚义平,陶俊,王宏,等.嵌合内含子对抗bFGF抗体基因在293T细胞中表达的影响.中国生物工程杂志,2010,30(3):9-14.
[16]Liu XL,Zhang W,Tang SJ.Intracranial transplantation of human adipose-derived stem cells promotes the expression of neurotrophic factors and nerve repair in rats of cerebral ischemia-reperfusion injury.Int J Clin Exp Pathol,2014,7(1):174-183.
[17]Li CJ,Ma YH,Yi KL,et al.The interactions between nerve growth factor and gonadotrophins in bovine oviduct.Animal Reprod Sci,2014,149(3/4):117-123.
[18]Recillas-Targa F,Pikaart MJ,Burgess-Beusse B,et al.Position-effect protection and enhancer blocking by the chickenβ-globin insulator are separable activities.Proc Natl Acad Sci USA,2002,99(10):6883-6888.
[19]Fath S,Bauer AP,Liss M,et al.Multiparameter RNAand codon optimization:a standardized tool to assess and enhance autologous mammalian gene expression.PLoS ONE,2011,6(3):e17596.
[20]Lin Y,Li ZX,Wang TY,et al.MAR characteristic motifs mediate episomal vector in CHO cells.Gene,2015,559(2):137-143.
[21]Wang F,Zhang JH,Wang TY,et al.Regulating effects of insertion direction of matrix attachment regions on transgenic expression in stably transformed Chinese hamster ovary cells.Genet Mol Res,2015,14(2):7031-7038.
[22]Xu L,Li YH,Shi XC,et al.Expression,purification,and characterization of recombinant mouse nerve growth factor in Chinese hamster ovary cells.Protein Expr Purif,2014,104:41-49.
[23]Wang XY,Zhang JH,Sun QL,et al.Characteristic element of matrix attachment region mediates vector attachment and enhances nerve growth factor expression in Chinese hamster ovary cells.Genet Mol Res,2015,14(3):9191-9199.