Departamento de Biotecnología, INIA, Madrid, Spain.
Alternative Gene Expression S.L. Pozuelo de Alarcón, Madrid, Spain.
J Virol Methods. 2017 Dec;250:17-24. doi: 10.1016/j.jviromet.2017.09.017. Epub 2017 Sep 22.
Trichoplusia ni insect larvae infected with vectors derived from the Autographa californica multiple nucleopolyhedrovirus (AcMNPV), are an excellent alternative to insect cells cultured in conventional bioreactors to produce recombinant proteins because productivity and cost-efficiency reasons. However, there is still a lot of work to do to reduce the manual procedures commonly required in this production platform that limit its scalability. To increase the scalability of this platform technology, a current bottleneck to be circumvented in the future is the need of injection for the inoculation of larvae with polyhedrin negative baculovirus vectors (Polh-) because of the lack of oral infectivity of these viruses, which are commonly used for production in insect cell cultures. In this work we have developed a straightforward alternative to obtain orally infective vectors derived from AcMNPV and expressing recombinant proteins that can be administered to the insect larvae (Trichoplusia ni) by feeding, formulated in the insect diet. The approach developed was based on the use of a recombinant polyhedrin protein expressed by a recombinant vector (Polh+), able to co-occlude any recombinant Polh- baculovirus vector expressing a recombinant protein. A second alternative was developed by the generation of a dual vector co-expressing the recombinant polyhedrin protein and the foreign gene of interest to obtain the occluded viruses. Additionally, by the incorporation of a reporter gene into the helper Polh+ vector, it was possible the follow-up visualization of the co-occluded viruses infection in insect larvae and will help to homogenize infection conditions. By using these methodologies, the production of recombinant proteins in per os infected larvae, without manual infection procedures, was very similar in yield to that obtained by manual injection of recombinant Polh- AcMNPV-based vectors expressing the same proteins. However, further analyses will be required for a detailed comparison of production yields reached by injection vs oral infections for different recombinant proteins. In conclusion, these results open the possibility of future industrial scaling-up production of recombinant proteins in insect larvae by reducing manual operations.
感染了源自美洲棉铃虫多角体病毒（AcMNPV）载体的斜纹夜蛾幼虫，是替代传统生物反应器中培养昆虫细胞生产重组蛋白的绝佳选择，因为从生产力和成本效益方面考虑，这种方法更具优势。然而，由于该生产平台通常需要大量的人工操作，限制了其可扩展性，因此仍有许多工作要做。为了提高该平台技术的可扩展性，未来需要解决的一个当前瓶颈是需要对幼虫进行注射，以便用多角体蛋白阴性杆状病毒载体（Polh-）进行接种，因为这些病毒缺乏口服感染力，常用于昆虫细胞培养的生产。在这项工作中，我们开发了一种简单的替代方法，可获得源自 AcMNPV 的口服感染性载体，并表达可通过喂食（在昆虫饲料中配制）施用于昆虫幼虫（斜纹夜蛾）的重组蛋白。所开发的方法基于使用重组载体表达的重组多角体蛋白（Polh+），该蛋白能够与表达重组蛋白的任何重组 Polh-杆状病毒载体共同包埋。另一种替代方法是通过生成共表达重组多角体蛋白和感兴趣的外源基因的双载体来获得包埋的病毒。此外，通过将报告基因插入辅助 Polh+载体中，可跟踪观察到受感染的幼虫中共同包埋的病毒的感染情况，有助于均匀化感染条件。通过使用这些方法，无需进行人工感染程序，通过经口感染幼虫可生产出与通过手动注射表达相同蛋白的重组 Polh- AcMNPV 基载体所获得的产量非常相似的重组蛋白。然而，对于不同的重组蛋白，需要进一步的分析来详细比较注射和口服感染的产量。总之，这些结果为通过减少人工操作来实现未来在昆虫幼虫中生产重组蛋白的工业规模扩大提供了可能性。
