The glycoprotein of infectious hematopoietic necrosis virus (IHNV), the causative agent of acute disease in salmonids, is the only structural protein of the virus that can induce protective immunity in the fish host. Here, the reliability of bean (Phaseolus vulgaris) plant for the production of this viral protein was examined by the transient expression method. Using the syringe agroinfiltration method, leaves of bean plants were transformed with the expression construct encoding the full-length of IHNV glycoprotein (IHNV-G) gene. Furthermore, the transformation efficacy of two infiltration buffers including PBS-A (PBS+acetosyringone) and MMS-A (MES buffer + MgSO₄ + sucrose + acetosyringone) was compared. The analysis of mRNA and dot-blot assay confirmed the transcription and translation of IHNV-G protein in bean leaves. Moreover, Western blotting verified the production of intact, full-length (∼57 kDa) IHNV-G protein in the agroinfiltrated plants. Of note, the production level of IHNV-G using MMS-A agroinfiltration buffer was approximately five times higher compared to PBS-A buffer (0.48 vs. 0.1% of total soluble protein), indicating the effect of infiltration buffer on the transient transformation efficiency. The recombinant protein was purified at the final yield of 0.35 μg/g of fresh leaf tissue, using nickel affinity chromatography. The present work is the first report describing the feasibility of the plant expression platform for the production of IHNV-G protein, which can be served as an oral vaccine against IHNV infection.

中文翻译：

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通过农杆菌浸润在豆（菜豆）叶中瞬时表达来自传染性造血坏死病毒的全长糖蛋白

传染性造血坏死病毒 (IHNV) 的糖蛋白是鲑鱼急性疾病的病原体，是该病毒唯一可以在鱼类宿主中诱导保护性免疫的结构蛋白。在这里，通过瞬时表达方法检查了豆 ( Phaseolus vulgaris ) 植物生产这种病毒蛋白的可靠性。使用注射器农杆菌渗透法，用编码全长 IHNV 糖蛋白 ( IHNV-G ) 基因的表达构建体转化豆类植物的叶子。此外，包括 PBS-A（PBS+乙酰丁香酮）和 MMS-A（MES 缓冲液 + MgSO ₄）在内的两种浸润缓冲液的转化功效 + 蔗糖 + 乙酰丁香酮）进行比较。mRNA和斑点印迹分析证实了IHNV-G蛋白在豆叶中的转录和翻译。此外，蛋白质印迹验证了农杆菌浸润植物中完整、全长（~57 kDa）IHNV-G 蛋白的产生。值得注意的是，与 PBS-A 缓冲液相比，使用 MMS-A 农杆菌浸润缓冲液的 IHNV-G 生产水平大约高出五倍（0.48 与 0.1% 的总可溶性蛋白），表明浸润缓冲液对瞬时转化效率的影响. 使用镍亲和层析纯化重组蛋白，最终产量为 0.35 μg/g 新鲜叶组织。目前的工作是描述植物表达平台用于生产 IHNV-G 蛋白的可行性的第一份报告，