石斑魚虹彩病毒主要外鞘蛋白質基因轉殖至萵苣之研究

Studies on the Gene of Major Capsid Protein of Grouper Iridovirus Transferred into Lettuce (Lactuca sativa L.)

石斑魚（Epinephelus spp.）為臺灣地區具有重要經濟價值之養殖水產品，近年來受到石斑魚虹彩病毒（grouper iridovirus, GIV）的侵害，造成極高的致死率。目前臺灣上市的石斑魚不活化疫苗經腹腔注射後能夠提供魚隻保護效力。但在頻繁爆發傳染疫情的情況下，需要更低成本且方便的疫苗策略。轉殖植物提供具成本效益與生產及運送病毒蛋白質的可行系統，且可能有當作口服疫苗的潛力。構成GIV外殼的主要外鞘蛋白質（major capsid protein, MCP）約占病毒總蛋白質之40%，在不同品系間具有高度保留性，是一個具潛力的抗原候選者。本研究使用之MCP基因係以CaMV35S啟動子驅使，利用農桿菌媒介轉殖法生產轉殖萵苣。轉殖株以即時定量反轉錄聚合酶連鎖反應（quantitative real-time reverse transcription polymerase chainreaction, qRT-PCR）偵測MCP基因的轉錄表現，再以西方轉漬分析確認MCP蛋白質的表現。根據酵素聯結免疫吸附分析（enzyme-linked immunosorbent assay, ELISA），MCP蛋白質在不同轉殖品系之葉片組織內，含量介於0.28-0.53μg+mg^(-1)總可溶性蛋白質。以轉殖萵苣萃取物進行小鼠皮下注射及口服免疫，可偵測到MCP專一性抗體。於小鼠免疫後顯示，脾臟中的IFN-γ及IL-10會向上調節。本研究結果建議萵苣是具有表現MCP蛋白質潛力的生物反應器，未來可進一步應用於飼料添加物，進行魚隻口服試驗，以證實其保護效力。 The production of groupers Epinephelus spp. is critical to the Taiwanese economy. Recently, the grouper iridovirus (GIV) infection has caused considerable fatality rates in groupers. A commercially available inactive vaccine administered through intraperitoneal injection currently provides effectives protection to groupers. However, to combat the frequent occurrences of the GIV epidemics, a more convenient and economical vaccine strategy is required. Transgenic plants provide cost-effective and scalable systems for the production and delivery of viral protein. These plants have potential to be need as oral vaccines. The major capsid protein (MCP) of GIV capsid accounts for approximately 40% of the total virus protein. Due to the high conservation among various races, the MCP is a potential antigen candidate. In this study, we generated a transgenic lettuce expressing the GIV MCP gene, under the control of the CaMV 35S promoter, by using Agrobacterium-mediated transformation. We confirmed the transcriptional expression of the MCP gene using quantitative real-time RT-PCR analysis. We further confirmed the MCP translation by using western blot analysis. Based on ELISA analysis, the amount of MCP in leaf tissues of different transgenic lines ranged between 0.28 to 0.53 μg．mg^(-1) of the total soluble protein. We detected MCP-specific antibodies in mice following subcutaneous and oral administration with protein extracts from transgenic lettuce. Following the vaccination with the recombinant MCP, we identified upregulated IFN-γ and IL-10 in splenocytes of the mice. The preliminary results showed that lettuce is a potential bioreactor for the expression of MCP gene. Future studies would apply the transgenic lettuce as additives for groupers to validate its effectiveness.