高表达WSSV VP28+VP19囊膜蛋白转基因盐藻株的制备及其免疫作用分析

White Spot Syndrome Virus (WSSV) is one of an important pathogen that adversely impacts shrimp aquaculture, and there is still no control method to it. Current research hotspot was focus on the fields of using the WSSV subunit vaccine to immunization shrimp to prevention WSSV. Under laboratory conditions, applied WSSV vaccines to immune to shrimp can generate the positively protective function. Shrimp are the natural hosts for WSSV, and the WSSV structural proteins can be introduced into Dunaliella salina (D. salina) cells by transformation; in turn, D. salina cells can be used as crude bait for shrimp. Based on these natural intrinsic interactions, this project attempted to prepare transgenic D. salina strains that as a live oral vaccine to immune shrimp come to producing the immune protection. Under the previous NSFC funded, the research show that approximately 70% protection rate can be achieved by using transgenic D. salina to immune shrimp. To enhance the protective effect of transgenic D. salina strains to shrimp, the prerequisite is to improving the expression of foreign genes in D. salina cells. Therefore, this project could be used some strategies to increasing the protein yields, including the clone of endogenous promoters, reconstruction of novel eukaryotic expression vector, expression of fused proteins, and exploitation of chloroplast expression system of D. salina. Preparation of high expression of WSSV envelope protein VP28+VP19 transgenic D. salina strains will accelerate emergence of effective vaccines, and provides a hope and bright prospects to early controlling the occurrence and development of WSSV.

白斑综合征病毒(WSSV)是对虾养殖业的重要病原体之一，目前对其仍无有效的防治方法。利用WSSV亚单位疫苗对虾体进行免疫防治成为当前的研究热点，在实验室条件下疫苗免疫虾体有着确切的保护作用。本课题借助于WSSV、盐藻和对虾三者之间存在的天然内在联系，即WSSV感染对虾、对虾以盐藻为饵料、而盐藻作为表达系统又能够表达病毒蛋白，通过基因工程手段制备表达病毒蛋白的转化藻株，将该藻株作为疫苗应用于对虾的免疫保护。前期资助课题研究表明，VP28转化藻株通过口服途径免疫虾体产生约70%的保护率。为提高转基因藻株对虾体的保护作用，提高外源基因在盐藻中的表达量是首要条件。本课题拟通过克隆内源性启动子、构建新型表达载体、表达融合蛋白和开发盐藻叶绿体系统等途径来提高外源基因的表达量。高表达WSSV VP28+VP19蛋白转基因藻株的制备将加快高效疫苗的问世，为尽早控制白斑病的发生与发展提供了曙光和美好前景。

针对于白斑综合征病毒的防控，疫苗的开发和应用成为了当前国内外的研究热点和主流方向。以盐藻细胞作为新型的表达系统，通过基因工程手段制备表达WSSV囊膜蛋白的转基因藻株，并以此藻株直接作为疫苗投喂虾体，能够取得显著的免疫保护效果。为进一步提高转基因藻株对虾体的保护作用，提高外源基因在盐藻中的表达量是首要条件。提高目的基因在盐藻中的表达量，本课题原设计路线有二，一是利用盐藻或WSSV内源性启动子和增强子等调控元件，重新构建盐藻表达载体，进行盐藻细胞核的转化；二是开发盐藻的另一套新型表达系统，即盐藻叶绿体表达系统的建立。按照课题的进度安排，课题组完成了各目标任务。课题的主要进展有：1. 利用内源性启动子重新构建了细胞核重组表达载体，并实现外源蛋白在盐藻细胞核的成功表达。动物免疫实验证实了转基因藻株有着约60%的免疫保护作用；2. 通过初步尝试，证明了外源基因能够被成功引入叶绿体系统，且该转化株对动物有着约65%的保护率。但是，无论是盐藻细胞核系统，或是盐藻叶绿体系统，均没有实现提高免疫保护效果的目的；3. 建立了转化盐藻细胞的自转化方法，该方法具有操作非常简便、简便快捷、省时省力、不需要额外试剂和仪器等优点，能够自发的将外源基因有效的导入盐藻细胞；4. 为查明影响疫苗保护效果的因素，全面统计分析了当前各种WSSV疫苗的效果现状，得到了疫苗种类、免疫方式、单价多价型疫苗、病毒亚单位蛋白等指标的优化结果，这为将来的课题深入研究工作提供了指导。