Development of a novel inducible Baculovirus-Insect cell expression system

1)目前基于加利福尼亚多核多角体病毒（AcMNPV）的杆状病毒-昆虫细胞表达系统被广泛应用于大规模蛋白生产。这包括生物和医学研究的各个领域，以及生产用于疫苗生产的高纯度蛋白质或工业过程中使用的酶。不幸的是，该系统的有效性和蛋白质产量往往受到所需蛋白质对实际蛋白质表达发生的昆虫细胞的毒性作用的限制。例如，这可能会导致整个细胞培养物的不完全感染，因为在感染的早期阶段病毒复制有限。因此，我们打算开发一种诱导表达系统，它可以阻止感兴趣的蛋白质的表达，同时允许病毒在细胞培养物中完全复制和传播。因此，在诱导蛋白表达的时间点上，所有细胞都被认为是均匀感染的，从而导致相当高的产量。2)此外，该诱导体系还可用于建立一种新的杆状病毒增强型生物农药及其成本效益生产。目前，各种昆虫病原病毒作为生物农药应用于经济相关植物上，但目前的杀虫效果主要是由于病毒在宿主细胞内大量复制并裂解而产生的。为了提高农药效率，可以在AcMNPV中加入额外的昆虫毒素，这一方法也被广泛应用于生物农药。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的复制，从而限制病毒颗粒的产量。这个问题可以通过为研究目的而开发的相同的诱导表达系统来解决。而在细胞培养和病毒复制的感染过程中，不会产生有毒蛋白，毒素的表达只能在田间触发，从而最大化了病毒的复制/生产。3)在第二种方法中，如果前面描述的诱导表达系统在AcMNPV中有效，我们建议将这一概念额外应用于相关的pomonella granulo病毒（CpGV）。由于现有的几个AcMNPV专利限制了成本效益的使用，并且也涵盖了改进的AcMNPV，因此迄今为止未申请的CpGV是一个合理的替代方案。综上所述，该项目的成果可用于各类分子/医学研究以及高产蛋白质生产和生物农药开发的商业领域。

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.