Development of a novel inducible Baculovirus-Insect cell expression system

新型诱导型杆状病毒-昆虫细胞表达系统的开发

• 批准号: 226535580
• 负责人: Professor Dr. Rainer Buchholz
• 金额: --
• 依托单位: Department of Applied Biology
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2012
• 资助国家: 德国
• 起止时间: 2011-12-31 至 2014-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/226535580?language=en
• 关键词: Development; novel; inducible; Baculovirus; Insect

1) Currently the Baculovirus-insect cell expression system based on the Autographa californica multinucleopolyhedro virus (AcMNPV) is widely applied to large scale protein production. This includes various fields of biological and medical research as well as the production of highly pure protein for vaccine production or of enzymes used in industrial processes.Unfortunately, effectiveness and protein yield of this system is quite often limited by toxic effects of the desired protein to the insect cells in which the actual protein expression takes place. This for example may cause an incomplete infection of the whole cell culture due to limited virus replication in already early infection stages. Therefore we intend to develop an inducible expression system which prevents expression of the protein of interest while allowing full replication and spreading of the virus within the cell culture. At the time point of induction of protein expression, all cells are therefore thought to be homogenously infected leading to a considerably higher yield.2) Moreover, this inducible system can also be applied to establish a new enhanced baculovirus-based bio-pesticide and its cost-efficient production. While currently various insect pathogenic viruses are being applied as bio-pesticides on economically relevant plants, up to now the insecticide effect is mainly caused by the overwhelming virus replication in host cells and their subsequent lysis. In order to increase pesticide efficiency, additional insect toxins could be incorporated in AcMNPV, a procedure which is also extensively applied as bio-pesticide. During large scale industrial production of such a modified virus in cell culture bioreactors, toxicity of the additional protein encoded in the viral genome would also limit virus replication and thus the yield of virus particles.This problem can be addressed by the very same inducible expression system as developed for research purposes. While during infection of the cell culture and replication of the virus, no toxic proteins are to be produced, the expression of the toxin could be triggered only on the field thus maximizing beforehand virus replication / production.3) In a second approach, we propose to apply this concept additionally to the related Cydia pomonella granulo virus (CpGV), if the beforehand described inducible expression system is effective in AcMNPV. As several already existing patents on AcMNPV are limiting cost effective usage and would also cover a modified AcMNPV, the up to now unclaimed CpGV is a reasonable alternative. In summary, the results of this project can be used in various classes of molecular / medical research as well as in the commercial sector for high yield protein production and bio pesticide development.

1)目前广泛应用于规模化蛋白质生产的是基于链球虫多核多角体病毒(AcMNPV)的杆状病毒昆虫细胞表达系统。这包括生物和医学研究的各个领域，以及用于疫苗生产或工业过程中使用的酶的高纯度蛋白质的生产。不幸的是，该系统的有效性和蛋白质产量经常受到所需蛋白质对实际蛋白质表达所在的昆虫细胞的毒性影响的限制。例如，这可能会导致整个细胞培养物的不完全感染，因为病毒复制在已经处于早期感染阶段。因此，我们打算开发一种可诱导的表达系统，以防止目的蛋白的表达，同时允许病毒在细胞培养中完全复制和传播。因此，在蛋白质表达诱导的时间点，所有细胞都被认为是均匀感染的，从而获得相当高的产量。2)此外，该诱导系统还可以用于建立新的基于杆状病毒的强化生物农药及其成本效益生产。虽然目前各种昆虫致病病毒被作为生物农药应用于经济相关的植物，但到目前为止，杀虫效果主要是由病毒在宿主细胞中的大量复制和随后的裂解引起的。为了提高农药效率，可以在AcMNPV中加入额外的昆虫毒素，这一方法也被广泛用作生物农药。在细胞培养生物反应器中大规模工业化生产这种修饰病毒时，病毒基因组中编码的额外蛋白质的毒性也会限制病毒复制，从而限制病毒颗粒的产量。这个问题可以通过为研究目的开发的完全相同的诱导表达系统来解决。虽然在细胞培养和病毒复制的感染过程中，不会产生有毒蛋白，但毒素的表达只能在田间被触发，从而使预先的病毒复制/生产最大化。3)在第二种方法中，如果先前描述的诱导表达系统在AcMNPV中有效，我们建议将这一概念另外应用于相关的Cydia pomonella grao病毒(CpGV)。由于AcMNPV上已有的几项专利限制了成本效益的使用，还将涵盖一种改进的AcMNPV，到目前为止，无人要求保护的CpGV是一个合理的替代方案。总而言之，该项目的成果可用于各种类型的分子/医学研究，以及用于高产蛋白质生产和生物农药开发的商业部门。