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构建高效的杆状病毒展示系统及其在多基因共展示中的应用。

Construction of a highly efficient display system for baculovirus and its application on multigene co-display.

机构信息

Guangdong Provincial Key Laboratory of Agro-Animal Genomics and Molecular Breeding and Subtropical Sericulture and Mulberry Resources Protection and Safety Engineering Research Center, College of Animal Science, South China Agricultural University, Guangzhou, 510642, Guangdong, China.

Guangzhou Cynosure Bio-Technology Co., Ltd, Guangzhou, 510080, Guangdong, China.

出版信息

Mol Genet Genomics. 2018 Oct;293(5):1265-1277. doi: 10.1007/s00438-018-1459-9. Epub 2018 Jun 19.

Abstract

The classical baculovirus display system (BDS) has often recruited fields including gene delivery, gene therapy, and the genetic engineering of vaccines, as it is capable of presenting foreign polypeptides on the membranes of recombinant baculovirus through a transmembrane protein. However, classical BDS's high cost, complicated operation, low display efficiency and its inability to simultaneously display multiple gene products impede its practicality. In this study, we present a novel and highly efficient display system based on ires-dependent gp64 for rescuing gp64-null Bacmid of baculovirus construction without affecting the viral replication cycle, which we name the baculovirus multigene display system (BMDS). Laser scanning confocal microscopy demonstrated that eGFP, eYFP, and mCherry were translocated on the membrane of Spodoptera frugiperda 9 cell successfully as expected. Western blot analysis further confirmed the presence of the fluorescent proteins on the budded, mature viral particles. The results showed the display efficiency of target gene on cell surface is fourfold that of classical BDS. In addition, a recombinant baculovirus displaying three kinds of fluorescent proteins simultaneously was constructed, thereby demonstrating the effectiveness of BMDS as a co-display system.

摘要

经典杆状病毒展示系统(BDS)常用于基因传递、基因治疗和疫苗的遗传工程等领域,因为它能够通过跨膜蛋白将外源多肽展示在重组杆状病毒的膜上。然而,经典 BDS 的高成本、复杂的操作、低展示效率以及无法同时展示多个基因产物的缺点,限制了其实际应用。在本研究中,我们提出了一种基于 IRES 依赖性 gp64 的新型高效展示系统,用于拯救杆状病毒构建中无 gp64 缺失的 Bacmid,而不影响病毒复制周期,我们将其命名为杆状病毒多基因展示系统(BMDS)。激光共聚焦显微镜显示,eGFP、eYFP 和 mCherry 如预期的那样成功转位到 Spodoptera frugiperda 9 细胞的膜上。Western blot 分析进一步证实了荧光蛋白存在于芽生的、成熟的病毒颗粒上。结果表明,目标基因在细胞表面的展示效率是经典 BDS 的四倍。此外,构建了一种同时展示三种荧光蛋白的重组杆状病毒,从而证明了 BMDS 作为一种共展示系统的有效性。

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