家蚕-杆状病毒表达载体系统(家蚕-BEVS),利用家蚕核型多角体病毒(BmNPV)和家蚕幼虫或蛹,已被用作生产各种重组蛋白的具有成本效益的表达系统。最近,杆状病毒中的几种基因敲除已被证明可以提高重组蛋白的生产率。然而,杆状病毒基因组的基因编辑(约130kb)仍然具有挑战性和耗时。在这项研究中,我们试图通过使用两步金门组装(GGA)从含有BmNPV基因组DNA片段的质粒中合成和基因编辑BmNPVbacmid,进一步提高家蚕BEVS的生产力。BmNPV基因组,分成19个片段,通过PCR扩增并克隆到质粒中。从这些初始质粒中,用IIS型限制酶BsaI通过GGA构建了四个含有BmNPV基因组DNA的中间质粒。随后,通过GGA与另一种IIS限制性酶PaqCI从四个中间质粒成功合成了全长杆粒,效率高达97.2%。此外,这种方法能够快速而直接地产生缺乏六个基因的BmNPVbacmid,抑制了蚕蛹中表达的重组蛋白的降解。这些结果表明,仅使用简单的克隆技术和酶促反应就可以快速有效地编辑BmNPVbacmid,标志着蚕BEVS的改进取得了重大进展。
The silkworm-baculovirus expression vector system (silkworm-BEVS), using Bombyx mori nucleopolyhedrovirus (BmNPV) and silkworm larvae or pupae, has been used as a cost-effective expression system for the production of various recombinant proteins. Recently, several gene knockouts in baculoviruses have been shown to improve the productivity of recombinant proteins. However, the gene editing of the baculovirus genome (approximately 130kb) remains challenging and time-consuming. In this study, we sought to further enhance the productivity of the silkworm-BEVS by synthesizing and gene editing the BmNPV bacmid from plasmids containing fragments of BmNPV genomic DNA using a two-step Golden Gate Assembly (GGA). The BmNPV genome, divided into 19 fragments, was amplified by PCR and cloned into the plasmids. From these initial plasmids, four intermediate plasmids containing the BmNPV genomic DNA were constructed by GGA with the type IIS restriction enzyme BsaI. Subsequently, the full-length bacmid was successfully synthesized from the four intermediate plasmids by GGA with another type IIS restriction enzyme PaqCI with a high efficiency of 97.2%. Furthermore, this methodology enabled the rapid and straightforward generation of the BmNPV bacmid lacking six genes, resulting in the suppression of degradation of recombinant proteins expressed in silkworm pupae. These results indicate that the BmNPV bacmid can be quickly and efficiently edited using only simple cloning techniques and enzymatic reactions, marking a significant advancement in the improvement of the silkworm-BEVS.