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Enhancing the Butyrylcholinesterase Activity in HEK-293 Cell Line by Dual-Promoter Vector Decorated on Lipofectamine.
Drug Design, Development and Therapy ( IF 4.8 ) Pub Date : 2020-09-04 , DOI: 10.2147/dddt.s260419 Vida Mirzaie 1 , Touba Eslaminejad 2 , Homayoon Babaei 3 , Seyed Noureddin Nematollahi-Mahani 4, 5
Drug Design, Development and Therapy ( IF 4.8 ) Pub Date : 2020-09-04 , DOI: 10.2147/dddt.s260419 Vida Mirzaie 1 , Touba Eslaminejad 2 , Homayoon Babaei 3 , Seyed Noureddin Nematollahi-Mahani 4, 5
Affiliation
Purpose: Human butyrylcholinesterase (BChE) serves as a bio scavenger to counteract organophosphate poisoning. It is also a potential drug candidate in several therapeutic fields. Therefore, in the present study, we constructed a new dual-promoter plasmid consisting of Cytomegalovirus (CMV) and human elongation factor 1α (EF-1α) promoters and transfected that into HEK-293 cells using Lipofectamine to enhance the BChE secretion.
Methods: The new dual-promoter construction (pBudCE dual BChE) including two copies of the BChE gene was designed and transfected into cells by liposomal structures. The cloned plasmids were evaluated by enzyme digestion and gel electrophoresis analysis. Experimental groups were categorized into the cells transfected by pBudCE dual BChE (treatment), pCMV (positive control) vectors, and nontransfected cells (negative control). BChE gene expression was evaluated by qRT-PCR and the enzyme activity was assessed using modified Ellman’s method. The freeze-thaw process was carried out for analyzing the stability of the pBudCE dual BChE vector.
Results: Validation examination of the cloned plasmids confirmed the successful cloning process. The gene expression level and Ellman’s method value in pBudCE dual BChE was higher than the other groups. CMV promoter has also increased the enzyme activity, although the difference was not significant compared with the control group. Interestingly, freeze-thaw cycles followed by several passages did not affect the enzyme activity.
Conclusion: The designed construction with CMV and EF-1α promoters could increase BChE gene expression and the activity of the BChE enzyme in HEK-293 cell line. Large-scale production of BChE enzyme can be achieved by using dual-promoter plasmid construction compared to a single-promoter vector to be used in clinical trials.
Keywords: dual-promoter, vector, recombinant protein, drug delivery, Ellman’s method, HEK-293
中文翻译：
通过修饰在 Lipofectamine 上的双启动子载体增强 HEK-293 细胞系中的丁酰胆碱酯酶活性。
目的：人丁酰胆碱酯酶 (BChE) 作为生物清除剂来对抗有机磷中毒。它也是几个治疗领域的潜在候选药物。因此，在本研究中，我们构建了由巨细胞病毒(CMV) 和人延伸因子 1α (EF-1α) 启动子组成的新双启动子质粒，并使用 Lipofectamine 将其转染到 HEK-293 细胞中以增强 BChE 分泌。
方法：新的双启动子构建（pBudCE dual BChE）包括两个BChE拷贝基因被设计并通过脂质体结构转染到细胞中。通过酶消化和凝胶电泳分析评估克隆的质粒。实验组分为转染pBudCE双BChE（治疗）的细胞、pCMV（阳性对照）载体和未转染的细胞（阴性对照）。通过 qRT-PCR 评估BChE基因表达，并使用改进的 Ellman 方法评估酶活性。进行冻融过程以分析 pBudCE 双 BChE 载体的稳定性。
结果：克隆质粒的验证检查证实了成功的克隆过程。pBudCE dual BChE的基因表达水平和Ellman法值均高于其他组。CMV启动子也增加了酶活性，尽管与对照组相比差异不显着。有趣的是，几代之后的冻融循环不影响酶活性。
结论： CMV和EF-1α启动子的设计构建可以提高HEK-293细胞系中BChE基因的表达和BChE酶的活性。与用于临床试验的单启动子载体相比，使用双启动子质粒构建可以实现 BChE 酶的大规模生产。
关键词：双启动子, 载体, 重组蛋白, 药物递送, 埃尔曼法, HEK-293
更新日期：2020-09-05
Methods: The new dual-promoter construction (pBudCE dual BChE) including two copies of the BChE gene was designed and transfected into cells by liposomal structures. The cloned plasmids were evaluated by enzyme digestion and gel electrophoresis analysis. Experimental groups were categorized into the cells transfected by pBudCE dual BChE (treatment), pCMV (positive control) vectors, and nontransfected cells (negative control). BChE gene expression was evaluated by qRT-PCR and the enzyme activity was assessed using modified Ellman’s method. The freeze-thaw process was carried out for analyzing the stability of the pBudCE dual BChE vector.
Results: Validation examination of the cloned plasmids confirmed the successful cloning process. The gene expression level and Ellman’s method value in pBudCE dual BChE was higher than the other groups. CMV promoter has also increased the enzyme activity, although the difference was not significant compared with the control group. Interestingly, freeze-thaw cycles followed by several passages did not affect the enzyme activity.
Conclusion: The designed construction with CMV and EF-1α promoters could increase BChE gene expression and the activity of the BChE enzyme in HEK-293 cell line. Large-scale production of BChE enzyme can be achieved by using dual-promoter plasmid construction compared to a single-promoter vector to be used in clinical trials.
Keywords: dual-promoter, vector, recombinant protein, drug delivery, Ellman’s method, HEK-293
中文翻译：
通过修饰在 Lipofectamine 上的双启动子载体增强 HEK-293 细胞系中的丁酰胆碱酯酶活性。
目的：人丁酰胆碱酯酶 (BChE) 作为生物清除剂来对抗有机磷中毒。它也是几个治疗领域的潜在候选药物。因此，在本研究中，我们构建了由巨细胞病毒(CMV) 和人延伸因子 1α (EF-1α) 启动子组成的新双启动子质粒，并使用 Lipofectamine 将其转染到 HEK-293 细胞中以增强 BChE 分泌。
方法：新的双启动子构建（pBudCE dual BChE）包括两个BChE拷贝基因被设计并通过脂质体结构转染到细胞中。通过酶消化和凝胶电泳分析评估克隆的质粒。实验组分为转染pBudCE双BChE（治疗）的细胞、pCMV（阳性对照）载体和未转染的细胞（阴性对照）。通过 qRT-PCR 评估BChE基因表达，并使用改进的 Ellman 方法评估酶活性。进行冻融过程以分析 pBudCE 双 BChE 载体的稳定性。
结果：克隆质粒的验证检查证实了成功的克隆过程。pBudCE dual BChE的基因表达水平和Ellman法值均高于其他组。CMV启动子也增加了酶活性，尽管与对照组相比差异不显着。有趣的是，几代之后的冻融循环不影响酶活性。
结论： CMV和EF-1α启动子的设计构建可以提高HEK-293细胞系中BChE基因的表达和BChE酶的活性。与用于临床试验的单启动子载体相比，使用双启动子质粒构建可以实现 BChE 酶的大规模生产。
关键词：双启动子, 载体, 重组蛋白, 药物递送, 埃尔曼法, HEK-293
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