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内含子方向对MAR表达载体重组CHO细胞转基因表达的调控作用

Effect of Intron Orientation on the Expression of Transgene Imposed by MAR Expression Vector in Stably Recombinant CHO Cells
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摘要 目的分析内含子方向对核基质结合区(matrix attachment region,MAR)表达载体在重组中国仓鼠卵巢(Chinese hamster ovary,CHO)细胞中对转基因表达水平的影响。方法 PCR扩增β-珠蛋白MAR,克隆至表达载体上,构建含MAR表达载体,将载体上一段内含子序列酶切正向、反向连接到载体上。酶切和测序鉴定正确后,转染中国仓鼠卵巢CHO细胞,G418筛选稳定转化的细胞株,ELISA分析氯霉素乙酰转移酶(chloramphenicol acetyltransferase,CAT)基因的表达水平。结果具有正向内含子的含MAR和不含MAR的表达载体CAT基因表达水平均高于含反向内含子的表达载体(P〈0.05),反向内含子存在情况下,MAR不能提高转基因表达。结论在重组CHO细胞中,内含子的方向影响转基因表达水平,正向内含子和MAR能提高转基因表达,反向内含子不能提高转基因表达水平。 Objective To determine the effect of intron orientation on the transgene expression level imposed by matrix attachment region(MAR)expression vector.Methods The MAR ofβ-globin was amplified by PCR,and then cloned into MAR expression vectors.An intron sequence was digested with restriction enzyme,ligated to the MAR expression vector in reverse orientation,and then transfected into Chinese hamster ovary(CHO)cells.The transfected stable cells were screened by G418.The level of chloramphenicol acetyltransferase(CAT)gene expression was analyzed by ELISA method.Results The transgene expression levels of CHO cells with the two expression vectors with a positive intron or without MAR were higher than that of CHO cells with an expression vector with reverse intron(P 0.05).MAR did not improve transgene expression with reverse intron presence.Conclusion Different orientation of intron can affect transgene expression in recombinant CHO cells.The transgene expression level can be increased using positive intron and MAR.
作者 李琴 赵春澎 王小引 孙秋丽 王天云 LI Qin , ZHAO Chun-peng , WANG Xiao-yin , SUN Qiu-li , WANG Tian-yun ( 1. Analysis & Test Laboratory, Xinxiang Medical University, Xinxiang 453003, China; 2. Department of Biochemistry and Molecular Biology, Xinxiang Medical University, Xinxiang 453003, China)
出处 《四川大学学报:医学版》 CAS CSCD 北大核心 2016年第2期189-191,243共4页 Journal of West China University of Medical Sciences
基金 国家自然科学基金(No.31371332)资助
关键词 核基质结合区 内含子 转基因表达 CHO细胞 Matrix attachment region Iintron Transgene expression CHO
作者简介 通讯作者,E-mail:wtianyuncn@126.com
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