應用熒光原位雜交技術檢測康柏西普基因在CHO細胞染色體上的整合狀態(tài)
發(fā)布時間：2019-04-03 10:42
【摘要】：CHO細胞是常用的哺乳動物表達工程細胞。外源基因整合至CHO細胞染色體后,在大規(guī)模蛋白質生產過程中,由于相關壓力撤除,外源基因存在丟失的可能,因此有必要對其整合穩(wěn)定性進行檢測�？蛋匚髌�(conbercept)是一個能夠特異性結合VEGF-A的各種異構體、VEGF-B以及PlGF,從而發(fā)揮抗血管生成活性的融合蛋白�？蛋匚髌漳壳耙言诿绹M入Ⅲ期臨床試驗。本文運用熒光原位雜交對康柏西普基因在CHO細胞的整合狀態(tài)進行了檢測,發(fā)現經過4和19次傳代后,康柏西普基因依然能穩(wěn)定整合在基因組上,并且呈現出3個特點:(1)分布在一條染色體上,而不是多條染色體上;(2)分布在較長的染色體上;(3)在同一染色體上有較多拷貝數。同時,熒光定量PCR結果證明基因拷貝數無明顯改變,ELISA檢測證明蛋白表達水平亦無明顯改變。上述實驗證明在經過19次傳代以后,康柏西普基因仍然穩(wěn)定整合在基因組中,并可活躍表達,為康柏西普大規(guī)模生產及產品質控提供了有力依據。
[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.
【作者單位】： 軍事醫(yī)學科學院生物工程研究所;成都康弘生物科技有限公司;
【分類號】：Q78
[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.
【作者單位】： 軍事醫(yī)學科學院生物工程研究所;成都康弘生物科技有限公司;
【分類號】：Q78
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