遗传 ›› 2017, Vol. 39 ›› Issue (4): 326-332.doi: 10.16288/j.yczz.16-335

• 研究报告 • 上一篇    下一篇

应用荧光原位杂交技术检测康柏西普基因在CHO细胞染色体上的整合状态

师明磊1(),赖维莉2(),易天红2,柯潇2,赵志虎1()   

  1. 1. 军事医学科学院生物工程研究所,北京 100071
    2. 成都康弘生物科技有限公司,成都 610037
  • 收稿日期:2016-10-08 修回日期:2017-01-11 出版日期:2017-04-20 发布日期:2017-02-10
  • 作者简介:师明磊,博士,助理研究员,研究方向：表观遗传学。E-mail: shiml79@126.com|赖维莉,硕士,专业方向：细胞生物学。E-mail: laiweili@cnkh.com, shiml79@126.com|赵志虎,博士,研究员,研究方向：表观遗传学. E-mail: zhaozh@nic.bmi.ac.cn

Characterization of the conbercept gene localization in DHFR- amplified CHO cells using fluorescence in situ hybridization

Minglei Shi1(),Weili lai2(),Tianhong Yi2,Xiao Ke2,Zhihu Zhao1()   

  1. 1. Beijing Institute of Biotechnology, Beijing 100071, China
    2. Chengdu Kanghong Biotechnology Co. Ltd., Chengdu 610037, China
  • Received:2016-10-08 Revised:2017-01-11 Online:2017-04-20 Published:2017-02-10

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摘要：

CHO细胞是常用的哺乳动物表达工程细胞。外源基因整合至CHO细胞染色体后,在大规模蛋白质生产过程中,由于相关压力撤除,外源基因存在丢失的可能,因此有必要对其整合稳定性进行检测。康柏西普(conbercept)是一个能够特异性结合VEGF-A的各种异构体、VEGF-B以及PlGF,从而发挥抗血管生成活性的融合蛋白。康柏西普目前已在美国进入Ⅲ期临床试验。本文运用荧光原位杂交对康柏西普基因在CHO细胞的整合状态进行了检测,发现经过4和19次传代后,康柏西普基因依然能稳定整合在基因组上,并且呈现出3个特点：(1)分布在一条染色体上,而不是多条染色体上;(2)分布在较长的染色体上;(3)在同一染色体上有较多拷贝数。同时,荧光定量PCR结果证明基因拷贝数无明显改变,ELISA检测证明蛋白表达水平亦无明显改变。上述实验证明在经过19次传代以后,康柏西普基因仍然稳定整合在基因组中,并可活跃表达,为康柏西普大规模生产及产品质控提供了有力依据。

关键词: 荧光原位杂交, 康柏西普, CHO工程细胞, 基因组整合

Abstract:

Chinese-hamster ovary (CHO) cells are most widely used for mammalian protein expression. After integration into the CHO genome, the exogenous gene may be lost in the process of large-scale protein production due to the removal of related selection pressures. Therefore, it is necessary to test its stability in the genome. Conbercept is a fusion protein that specifically binds to the various isoforms of vascular endothelial growth factor (VEGF)-A, VEGF-B, and placental growth factor (PlGF), thereby exerting anti-angiogenic activities. It has been approved for Phase III clinical trials in the United States. In this study, fluorescence in situ hybridization (FISH) was used to localize the conbercept gene in dihydrofolatereductase (DHFR)-amplified CHO cell lines. Metaphase FISH showed that genomic integration of the conbercept gene was stable after 4 and 19 passages, and manifested three characteristics: first, the gene locates on one chromosome, rather than a number of chromosomes; second, the gene locates on the longer chromosomes; third, there are many copies located on the same chromosome. At the same time, the copy number of the conbercept gene in the CHO genome and the conbercept protein expression levels are also stable, as verified by qPCR and ELISA assays, respectively. These experiments demonstrated that the conbercept gene remained stable in the genome after 19 passages, and could be actively expressed, which strongly support the mass production and the quality control of conbercept.

Key words: fluorescence in situ hybridization (FISH), conbercept, CHO cell, chromosome integration