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

发布时间：2019-04-03 10:42

【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。
[Abstract]:CHO cells are commonly used mammalian expression engineering cells. After the foreign gene is integrated into the CHO cell chromosome, in the process of large-scale protein production, due to the removal of the related pressure, the possibility of loss of the foreign gene is possible, and therefore it is necessary to detect the whole stability. Conbercept is a fusion protein capable of specifically binding to various isomers of VEGF-A, VEGF-B and PlGF, thereby exerting anti-angiogenic activity. Cumbercept is currently in the U. S. for Phase III clinical trials. In this paper, we used the fluorescence in situ hybridization to detect the whole state of the conifer gene in CHO cells. After 4 and 19 passages, the Kopsipu gene can be stably integrated on the genome, and has three characteristics: (1) the distribution is on a chromosome, (2) on a longer chromosome; (3) a more copy number on the same chromosome. At the same time, the fluorescence quantitative PCR results showed no significant change in the copy number of the gene, and the ELISA test showed no change in the expression level of the protein. The above-mentioned experiments show that, after the 19 passages, the conifer gene is still stably integrated in the genome, and can be expressed actively, which provides a powerful basis for the mass production and quality control of the Cumbercept.
【作者单位】：军事医学科学院生物工程研究所;成都康弘生物科技有限公司;
【分类号】：Q78

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