摘要
目的研究人β-珠蛋白核基质附着区是否能够介导表达载体稳定附着于中国仓鼠卵巢(CHO)细胞。方法人β-珠蛋白MAR序列克隆到pEGFP-C1构建重组质粒载体pEGFP-MAR,转染CHO细胞,使用Hirt裂解法提取细胞中附着体质粒,CaCl2法转化附着体质粒到宿主大肠杆菌DH5ɑ中,提取质粒,酶切,测序分析。结果还原质粒双酶切和单酶切结果均与转染前质粒酶切结果一致;测序分析得知还原质粒与转染前质粒中插入片段DNA序列相同。结论人β-珠蛋白MAR序列重组载体在CHO细胞中以附着体形式被完整还原出来。还原质粒酶切结果和测序结果均与转染前质粒一致。
Objective To study the human β-globin matrix attachment region（MAR） sequence mediated vector wheth- er exist as stable episomes in transfected Chinese hamster ovary（CHO） cells. Methods Recombinant plasmid vector pEGFP- MAR was constructed by thrusting the amplified human β-globin MAR sequence into the parental pEGFP-C1. CHO cells were transfected both with pEGFP-C1 and reconstructed vector. Episomal vectors from the stable monoclonal cell lines were extracted by modificated Hirt cracking protocol. Then CaC12 method was performed to transform episomal vectors into the host bacteria E. coli DHSα,and the plasmid from the transformed E. coli DHSα was extracted. In the end, enzyme digestion identification and sequencing analysis were performed to detect the rescued plasmid and the original plasmid. Results Both double enzyme and single enzyme to completely digested the rescued plasmids and the original plassmid showed the same result. The sequencing a- nalysis showed that the inserted sequence of rescued plasmid was identical to the original DNA sequence. Conclusion Human 13-globin MAR characteristic sequence-based vector can be completely rescued from cultured CHO cells. The results of enzyme digestion identification and sequencing analysis are consistent in the rescued plasmids and the original plasmid.
出处
《新乡医学院学报》
CAS
2013年第9期695-699,共5页
Journal of Xinxiang Medical University
基金
河南省科技厅科技攻关计划资助项目(编号:112102310208)
新乡医学院研究生科研创新支持计划资助项目(编号:YJSCX201101Z)
