以神經壞死病毒類病毒顆粒作為石斑魚IL-6重組蛋白之奈米載體

Abstract

神經壞死病毒（Nervous Necrosis Virus, NNV）是一種高致病性的病毒，會導致病毒性神經壞死症（Viral Nervous Necrosis, VNN），對水產養殖業，特別是魚類幼苗造成嚴重的經濟損失。NNV的殼體為T=3二十面體結構，由RNA2所編碼的180個次單位殼蛋白組成，在病毒的穩定性與感染力中扮演關鍵角色。利用其自我組裝成T=3二十面體結構之特性，我們開發了NNV病毒樣顆粒（Virus-Like Particles, VLPs）作為攜帶重組石斑魚介白素-6（Recombinant Grouper Interleukine-6; rgIL-6）的奈米載體，rgIL-6對於對石斑魚幼苗免疫調節來說，是一個相當重要的細胞激素。奈米包覆的目的，希望能夠降低細胞激素傳遞時受到環境的影響，以及組織障壁的阻隔。首先必須將rgIL-6成功報復在NNV殼蛋白之中。我們先使用EGTA解聚緩衝液將NNV殼體蛋白解離為單體，接著再利用含二價金屬離子（如鈣離子）的重組緩衝液，在rgIL-6的存在下重新組裝回二十面體結構。這一個策略成功地實現了rgIL-6的包覆，並可以計算出重組蛋白的被包覆率。在適當的條件下，rgIL-6的被包覆效率可達55.67%（w/w），對應於約5:4的分子質量比例（NNV殼蛋白對rgIL-6）。此外，包覆於具蛋白酶耐受性的NNV VLPs內，rgIL-6可能免受酶解，進一步提升其穩定性與免疫刺激功能。與目前廣泛應用於人類醫學但成本高昂的脂質奈米粒子（Lipid Nanoparticles, LNPs）相比，NNV VLP是一種可規模化生產且具生物相容性的替代方案。先前的研究已證實，NNV VLPs可作為DNA疫苗質體的遞送載體，其具有進一步的應用的潛力。我們的研究結果顯示，與其他常見的二十面體結構VLPs相較，NNV VLPs作為細胞激素奈米載體展現出不錯的包覆效率。本研究針對NNV殼蛋白的單體解離與重組條件進行了優化，確保成功封裝rgIL-6，這個結果有望應用於石斑魚苗水產養殖產業，為功能性重組蛋白，特別是免疫促進劑的投遞策略提供一項新選擇。

Nervous Necrosis Virus (NNV) is a highly pathogenic virus responsible for viral nervous necrosis (VNN), causing significant economic losses in aquaculture, particularly among fish larvae. The NNV capsid, an icosahedral T=3 structure composed of 180 capsid protein subunits encoded by RNA2, plays a crucial role in viral stability and infectivity. Leveraging its self-assembling properties, we developed NNV Virus-Like Particles (VLPs) as nanocarriers for recombinant grouper interleukin-6 (rgIL-6), a cytokine critical for immune modulation in grouper larvae. To optimize encapsulation, we employed an EGTA-based disassembly buffer to break down the capsid into monomers, followed by a calcium-rich assembly buffer to restore its icosahedral structure in the presence of rgIL-6. This strategy successfully encapsulated rgIL-6 with an efficiency of 55.67% by mass, corresponding to a molecular ratio of approximately 5:4 (capsid protein to rgIL-6). Encapsulation within protease-resistant NNV VLPs protects rgIL-6 from enzymatic degradation, potentially enhancing its stability and immunostimulatory function. Unlike lipid nanoparticles (LNPs), which are widely used in human medicine but cost-prohibitive for aquaculture applications, NNV VLPs offer a scalable and biocompatible alternative. Previous studies with NNV VLPs have demonstrated the efficacy of VLPs in delivering DNA vaccine plasmid for cellular delivery, further reinforcing the versatility of this approach. Our findings highlight the potential of NNV VLPs as a cytokine-based nanocarrier platform, addressing key challenges in antigen stability and immune stimulation in aquaculture. Nervous Necrosis Virus (NNV), a highly pathogenic virus causing viral nervous necrosis (VNN), poses significant economic threats to aquaculture due to its devastating impact on fish species, particularly larvae. The capsid of NNV has been reported to adopt a T=3 icosahedral architecture, consisting of 180 capsid protein subunits encoded by RNA2, which contributes to viral stability and infectivity. Building upon this structural framework, we engineered virus-like particles (VLPs) from NNV capsid proteins as nanocarriers for recombinant grouper interleukin-6 (rgIL-6), a cytokine essential for immune modulation in grouper larvae. The study addressed key challenges in capsid protein disassembly and reassembly, employing EGTA-based disassembly buffers to break down the capsid into monomers and calcium-rich assembly buffers to restore its icosahedral architecture. Optimization of conditions ensured successful reassembly and encapsulation of rgIL-6. The encapsulation efficiency was optimized by determining the ideal ratio of capsid proteins to rgIL-6, resulting in VLPs that encapsulated rgIL-6 with an efficiency of 55.67% by mass, corresponding to a molecular ratio of approximately 5:4 (capsid protein to rgIL-6). Encapsulation within protease-resistant NNV VLPs may theoretically protect rgIL-6 from enzymatic degradation and enhance its delivery potential in clinical use. Leveraging this approach could pave the way for developing cytokine-based nanocarrier delivery methods for grouper aquaculture.