Effects of different 2A peptides on transgene expression mediated by tricistronic vectors in transfected CHO cells
Effects of different 2A peptides on transgene expression mediated by tricistronic vectors in transfected CHO cells
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不同2A肽对转染CHO细胞中三顺反子载体介导的转基因表达的影响
DOI：
10.1007/s11033-019-05153-3
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发表时间：
2019-10
影响因子：
2.8
通讯作者：
Dou Yuan-yuan
中科院分区：
文献类型：
--
作者：
Li Yan-mei;Wang Meng;Wang Tian-yun;Wei Yong-ge;Guo Xiao;Mi Chun-liu;Zhao Chun-peng;Cao Xiang-xiang;Dou Yuan-yuan
在中国仓鼠卵巢（CHO）细胞表达系统中，多顺反子载体可以增加转基因表达，减少基因表达不平衡。小的、自裂的2A肽具有很高的切割效率，对于构建高表达的多顺反电子载体至关重要。在这项研究中，我们研究了两种不同的2A肽通过对三反子载体的介导作用对CHO细胞转基因表达的影响。增强的绿色荧光蛋白（eGFP）和红色荧光蛋白（RFP）基因通过猪teschovirus-1 （P2A）和thesea asigna病毒（T2A）多肽在多顺反电子载体上连接。我们用这些载体转染CHO细胞，并筛选抗囊胚杀虫素菌落的存在。流式细胞术和实时定量PCR （qPCR）检测稳定转染细胞的eGFP和RFP表达水平及拷贝数。结果表明，与T2A相比，P2A可使eGFP和RFP的表达分别提高1.48倍和1.47倍。基因的表达水平与其拷贝数不成正比。综上所述，我们发现P2A可以有效地驱动CHO细胞中的转基因表达，并且一个有效的2A肽可以用于CHO细胞系统中重组蛋白的生产。
Multicistronic vectors can increase transgene expression and decrease the imbalance of gene expression in the Chinese hamster ovary (CHO) cell expression system. Small, self-cleaving 2A peptides have a high cleavage efficiency and are essential for constructing high-expression multicistronic vectors. In this study, we investigated the effects of two different 2A peptides on transgene expression in CHO cells via their mediating action on tricistronic vectors. The enhanced green fluorescent protein (eGFP) and red fluorescent protein (RFP) genes were linked by the porcine teschovirus-1 (P2A) and Thosea asigna virus (T2A) peptides in a multicistronic vector. We transfected CHO cells with these vectors and screened for the presence of blasticidin-resistant colonies. Flow cytometry and real-time quantitative PCR (qPCR) were used to detect the expression levels of eGFP and RFP and the copy numbers of stably transfected cells. The results showed that P2A could enhance eGFP and RFP expression by 1.48- and 1.47-fold, respectively, compared to T2A. The expression levels of the genes were not proportional to their copy numbers. In conclusion, we found that P2A can effectively drive transgene expression in CHO cells and a potent 2A peptide can be used for recombinant protein production in the CHO cell system.
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影响因子：
3.7
作者：
Ho SC;Bardor M;Li B;Lee JJ;Song Z;Tong YW;Goh LT;Yang Y
通讯作者：
Yang Y
影响因子：
3.7
作者：
Kim JH;Lee SR;Li LH;Park HJ;Park JH;Lee KY;Kim MK;Shin BA;Choi SY
通讯作者：
Choi SY
DOI：
10.1002/1521-2254(200007/08)2:4
发表时间：
2000-07
期刊：
The Journal of Gene Medicine
影响因子：
--
作者：
C. Rudolph;J. Lausier;S. Naundorf;R. Müller;J. Rosenecker
通讯作者：
C. Rudolph;J. Lausier;S. Naundorf;R. Müller;J. Rosenecker
影响因子：
1.6
作者：
Mariati;Ho, Steven C. L.;Yang, Yuansheng
通讯作者：
Yang, Yuansheng
基质附着区域组合增加转染的中国仓鼠卵巢细胞中的转基因表达
影响因子：
4.6
作者：
Zhao CP;Guo X;Chen SJ;Li CZ;Yang Y;Zhang JH;Chen SN;Jia YL;Wang TY
通讯作者：
Wang TY