Development and optimization of a pepino mosaic virus-based vector for rapid expression of heterologous proteins in plants,Applied Microbiology and Biotechnology

当前位置： X-MOL 学术 › Appl. Microbiol. Biotechnol. › 论文详情

Our official English website, www.x-mol.net, welcomes your feedback! (Note: you will need to create a separate account there.)

Development and optimization of a pepino mosaic virus-based vector for rapid expression of heterologous proteins in plants
Applied Microbiology and Biotechnology ( IF 3.9 ) Pub Date : 2021-01-04 , DOI: 10.1007/s00253-020-11066-0
Peter Abrahamian ₁ , John Hammond ₂ , Rosemarie W Hammond ₁

Affiliation

Abstract

Plant-virus–derived vectors are versatile tools with multiple applications in agricultural and medical biotechnology. In this study, we developed pepino mosaic virus (PepMV) (family Alphaflexiviridae; genus Potexvirus) into a vector for heterologous protein expression in plants. PepMV was initially cloned in a step-wise manner, fully sequenced and the full-length infectious clone was tested for infectivity in Nicotiana benthamiana. Initial infectious clones resulted in poor replication of PepMV and lack of systemic movement. Mutations in the viral sequence affected systemic infection. Two suspected mutations were altered to restore systemic infectivity. PepMV infection was apparent as early as 4 days post agroinfiltration (dpa) inoculation in N. benthamiana. A multiple cloning site was inserted into the PepMV genome for introduction and expression of foreign genes. Several modifications to the wild-type vector were made, such as a replacing the native subgenomic promoter (SGP) with a heterologous SGP, and introduction of translational enhancers and terminators, to improve heterologous expression of the foreign gene-of-interest. GFP was used as a reporter for monitoring virus infection and protein production. Strong GFP expression was observed as early as 4 dpa with a translational enhancer. The PepMV-based vector produces rapid expression of the foreign gene in comparison to two other potexvirus-based vectors. GFP production was monitored over time and optimal protein production was recorded between 5 and 7 dpa. GFP protein levels reached up to 4% and decreased to 0.5% total soluble protein at 7 and 14 dpa, respectively. Future studies will evaluate this virus-based vector for large-scale production of pharmaceutical compounds.

Key points

• A pepino mosaic virus isolate was developed into a plant-based expression vector.

• Expression levels of the heterologous protein were comparable or exceeded previously developed viral vectors.

• Protein levels in plants were highest between 5 and 7 days and decreased gradually.

Graphical abstract

中文翻译：

基于菠萝花叶病毒的载体的开发和优化，用于在植物中快速表达异源蛋白

抽象的

植物病毒衍生载体是多功能工具，在农业和医学生物技术中有多种应用。在这项研究中，我们将菠菜花叶病毒 (PepMV)（ Alphaflexiviridae科； Potexvirus属）开发成一种在植物中表达异源蛋白的载体。 PepMV 最初以逐步方式克隆、完全测序，并测试全长感染性克隆在本塞姆氏烟草中的感染性。最初的感染性克隆导致 PepMV 复制不良且缺乏全身运动。病毒序列突变影响全身感染。两个可疑的突变被改变以恢复全身感染性。早在本塞姆氏烟草中农杆菌渗透（dpa）接种后 4 天，PepMV 感染就很明显。将多克隆位点插入 PepMV 基因组中以引入和表达外源基因。对野生型载体进行了一些修饰，例如用异源 SGP 替换天然亚基因组启动子 (SGP)，以及引入翻译增强子和终止子，以改善外源目的基因的异源表达。 GFP 被用作监测病毒感染和蛋白质生产的报告基因。早在 4 dpa 时就用翻译增强子观察到强 GFP 表达。与其他两种基于 Potexvirus 的载体相比，基于 PepMV 的载体可快速表达外源基因。随着时间的推移监测 GFP 产量，并在 5 至 7 dpa 之间记录最佳蛋白质产量。 7 dpa 和 14 dpa 时，GFP 蛋白水平分别达到 4% 和下降至 0.5% 总可溶性蛋白。未来的研究将评估这种基于病毒的载体用于大规模生产药物化合物。