核基质附着区调控CHO细胞转基因表达序列分子特征的研究
发布时间：2019-06-19 17:57
【摘要】：背景利用基因重组技术可生产具有治疗作用的重组蛋白。针对重组蛋白的生产,目前应用最多的细胞表达系统是中国仓鼠卵巢(Chinese hamster ovary,CHO)细胞表达系统。但是CHO细胞表达系统存在转基因表达不稳定,表达水平较低等问题。前期研究发现核基质附着区(matrix attachment region,MAR)能克服转基因沉默,提高转基因的表达水平。然而,MAR调控转基因表达的机制、关键元件尚不清楚。目的分析核基质附着区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。方法根据NCBI GeneBank中人β-珠蛋白MAR序列,将人β-珠蛋白MAR全长共分为大小几乎相等的6段进行PCR克隆。设计PCR引物,引物的两端引入Kpn I和Bgl II酶切位点。以人全血基因组为模板,利用PCR技术依次从β-珠蛋白MAR的5′和3′端分段渐次克隆MAR各片段,片段之间相互重叠120 bp。将克隆好的MAR片段进行Kpn I和Bgl II双酶切,连接到载体pCATG启动子的上游,转化大肠杆菌DH5α感受态细菌,构建重组质粒pCAM1-6。将重组质粒pCAM1-6载体、pCAM和pCATG空载体转染CHO细胞,G418加压选择筛选,获得稳定转染CHO细胞克隆株,用ELISA方法检测不同稳定转染组中氯霉素乙酰转移酶(chloramphenicol acetyltransferase,CAT)的表达量,生物信息学分析各片段MAR序列特征。结果结果表明,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最显著。对MAR-1~MAR-6进行生物信息学分析发现:MAR-2含有1个MAR-like motif(AATATATTT);MAR-3含有1个MAR-like motif(AATATATTT)、2个Topo II位点;MAR-6含有1个Alu保守序列及β-globin cluster。结论MAR元件能够提高哺乳动物细胞中转基因的表达;MAR元件中MAR-like motif有助于转基因表达的提高。
[Abstract]:Background of the invention recombinant proteins having therapeutic effects can be produced using a gene recombination technique. For the production of recombinant protein, the most recently used cell expression system is Chinese Hamster Ovary (CHO) cell expression system. However, the expression system of CHO cells has the problems of unstable transgene expression and low expression level. The preliminary study found that the matrix attachment region (MAR) can overcome the transgenic silencing and improve the expression level of the transgene. However, MAR controls the mechanism of the transgene expression, and the key elements are not yet clear. Objective To study the molecular sequence characteristics of the gene expression in the nuclear matrix attachment region, and to identify the characteristic elements of MAR which can effectively improve the expression of the CHO cells. Methods According to the sequence of human-globin MAR in NCBI GeneBank, the full length of human-globin MAR was divided into 6 segments with almost the same size to carry out PCR cloning. PCR primers were designed and both ends of the primer were introduced into the Kpn I and Bgl II cleavage sites. By using the human whole blood genome as a template, the fragment of MAR was cloned from the 5-and 3-end of the MAR-globin MAR by PCR, and the fragments were overlapped with each other by 120 bp. The cloned MAR fragment was digested with Kpn I and Bgl II and ligated to the upstream of the vector pCATG promoter to transform the competent bacteria of E. coli DH5 to construct the recombinant plasmid pCAM1-6. The recombinant plasmid pCAM1-6 vector, pCAM and pCATG empty vector were transfected into CHO cells and G418 was selected for selection, and the CHO cell clones were stably transfected. The characteristics of MAR sequences were analyzed by bioinformatics. The results showed that the full length of the MAR-globin MAR could significantly improve the expression of the transgene, and the expression of the transgenic expression was most significant in the second segment of 421-1 020 and the third segment of 901-1,500. The bioinformatics analysis of MAR-1-MAR-6 found that MAR-2 contains 1 MAR-like motif (AATATTT), MAR-3 contains 1 MAR-like motif (AATATTT),2 Topo II sites, and MAR-6 contains 1 Alu conserved sequence and 1-global cluster. Conclusion MAR elements can improve the expression of transgenes in mammalian cells, and MAR-like motif in MAR elements contributes to the improvement of transgene expression.
【学位授予单位】：新乡医学院
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：Q78
本文编号：2502538
[Abstract]:Background of the invention recombinant proteins having therapeutic effects can be produced using a gene recombination technique. For the production of recombinant protein, the most recently used cell expression system is Chinese Hamster Ovary (CHO) cell expression system. However, the expression system of CHO cells has the problems of unstable transgene expression and low expression level. The preliminary study found that the matrix attachment region (MAR) can overcome the transgenic silencing and improve the expression level of the transgene. However, MAR controls the mechanism of the transgene expression, and the key elements are not yet clear. Objective To study the molecular sequence characteristics of the gene expression in the nuclear matrix attachment region, and to identify the characteristic elements of MAR which can effectively improve the expression of the CHO cells. Methods According to the sequence of human-globin MAR in NCBI GeneBank, the full length of human-globin MAR was divided into 6 segments with almost the same size to carry out PCR cloning. PCR primers were designed and both ends of the primer were introduced into the Kpn I and Bgl II cleavage sites. By using the human whole blood genome as a template, the fragment of MAR was cloned from the 5-and 3-end of the MAR-globin MAR by PCR, and the fragments were overlapped with each other by 120 bp. The cloned MAR fragment was digested with Kpn I and Bgl II and ligated to the upstream of the vector pCATG promoter to transform the competent bacteria of E. coli DH5 to construct the recombinant plasmid pCAM1-6. The recombinant plasmid pCAM1-6 vector, pCAM and pCATG empty vector were transfected into CHO cells and G418 was selected for selection, and the CHO cell clones were stably transfected. The characteristics of MAR sequences were analyzed by bioinformatics. The results showed that the full length of the MAR-globin MAR could significantly improve the expression of the transgene, and the expression of the transgenic expression was most significant in the second segment of 421-1 020 and the third segment of 901-1,500. The bioinformatics analysis of MAR-1-MAR-6 found that MAR-2 contains 1 MAR-like motif (AATATTT), MAR-3 contains 1 MAR-like motif (AATATTT),2 Topo II sites, and MAR-6 contains 1 Alu conserved sequence and 1-global cluster. Conclusion MAR elements can improve the expression of transgenes in mammalian cells, and MAR-like motif in MAR elements contributes to the improvement of transgene expression.
【学位授予单位】：新乡医学院
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：Q78
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