摘要
分析核基质结合区（matrix attachment region,MAR）调控转基因表达的分子序列特征,鉴定能有效提高CHO细胞转基因表达的MAR特征性元件。将人β-珠蛋白MAR片段从5＇到3＇端分为6个分段（1-540,421-1 020,901-1 500,1 381-1 980,1 861-2 460,2 341-2 999位置）,分别采用PCR进行克隆,经测序证实正确后,分别连接到含有氯霉素乙酰转移酶（chloramphenicol acetyltransferase,CAT）报告基因的表达载体SV40启动子及上游,构建β-珠蛋白MAR渐次片段介导的表达载体,转染CHO细胞,G418筛选出稳定表达细胞株,ELISA分析CAT报告基因的表达水平,生物信息学分析MAR序列特征。结果表明,β-珠蛋白MAR全长能显著提高转基因的表达,6个渐次片段相比较,421-1 020位的第2个分段和901-1 500位的第3个分段提高转基因表达作用显著。生物信息学分析结果显示,MAR-like motif有助于转基因表达提高。
The aim of this study was to analyze the molecular characteristics and identify the key elements of matrix attachment regions（ MARs） that can improve transgene expression in stably transfected CHO cells. β-globin MAR was divided into six sub-fragments（ position 1 - 540,421 - 1 020,901 - 1 500,1 381 - 1 980,1 861 - 2 460,2 341 - 2 999） from 5＇ to 3＇ end. These sub-fragments were gradually respectively cloned via PCR technology,and then inserted into upstream of SV40 promoter of expression vector including chloramphenicol acetyltransferase（ CAT） reporter gene expression cassette,followed by transfection into CHO cells and screened under G418. The CAT reporter gene expression was analyzed by ELISA. The sequence characteristic of each fragment was analyzed by bioinformatics. The results showed that the overall length of β-globin MAR can significantly improve the expression level of CAT. The second and third sub-fragments（ position 421 - 1 020,901 - 1 500） showed the most effective compared to other sub-fragments. The bioinformatics analysis results showed that MAR-like motif of MAR may contribute to the upregulation of transgene expression.
出处
《中国生物化学与分子生物学报》
CAS
CSCD
北大核心
2016年第1期64-68,共5页
Chinese Journal of Biochemistry and Molecular Biology
基金
国家自然科学基金项目(No.31371332)
研究生科研创新支持计划项目(No.YJSCX20428Y)资助~~
