基于杀鱼爱德华氏菌载体的大菱鲆圆环病毒疫苗构建及免疫效果评价

Development and Efficacy Evaluation of a Turbot Circovirus Vaccine Using an Edwardsiella piscicida Vector

摘要:

大菱鲆圆环病毒(Turbot circovirus, TCV)是近年来新发现且严重危害大菱鲆产业的重要病原。本团队前期已构建杀鱼爱德华氏菌(Edwardsiella piscicida)弱毒疫苗载体，在此基础上，本研究利用弱毒疫苗载体，针对TCV开展重组载体疫苗研发。通过对TCV衣壳蛋白(Cap)进行生物信息学分析，构建2种不同长度的Cap蛋白表达片段并克隆至质粒pYA3342，以缺失purA、asd双基因的杀鱼爱德华氏菌(LSE40ΔasdΔpurA)作为载体构建重组载体疫苗。Western Blot检测表明，Cap蛋白可以在重组载体中稳定表达。免疫实验结果显示，重组载体疫苗免疫大菱鲆对Cap蛋白的血清抗体效价显著上升；与对照组相比，实验组TCV病毒载量呈明显下降趋势。攻毒实验结果显示，两种重组载体疫苗对野生型杀鱼爱德华氏菌的免疫保护率分别为14.81%和62.97%。上述结果表明，本研究构建的重组载体疫苗能够有效诱导大菱鲆产生针对TCV的特异性体液免疫应答并能够抑制体内病毒复制，同时对杀鱼爱德华氏菌具有良好的免疫保护效果，展现出作为防控两种病原候选疫苗的潜力。

Abstract:

Turbot (Scophthalmus maximus) is an economically important marine fish species in the northern mariculture industry of China, with its farming areas extensively covering the Liaodong Peninsula and Shandong Peninsula, and it has gradually developed into one of the main industries of marine fish culture in northern China. However, with the continuous expansion of turbot farming scale and the increasing intensification, various disease problems have become increasingly prominent, gradually becoming a key bottleneck factor restricting the healthy and sustainable development of this industry. During the turbot farming process, Edwardsiellosis caused byEdwardsiella piscicidaand acute hemorrhagic disease caused by Turbot circovirus (TCV) are the two most severe disease types. The diseases caused by these two pathogens are characterized by acute onset, rapid transmission, and high mortality, causing serious economic losses to aquaculture practitioners. From the perspective of current prevention and control technology, the main intervention measures againstE. piscicidainfection are still antibiotic treatment and vaccination. However, the long-term and non-standard use of antimicrobial drugs in clinical settings has led to the increasingly prominent problem of pathogen resistance, which not only significantly reduces the therapeutic effect but also poses a potential threat to the safety of the aquaculture water environment and ecosystem. In terms of vaccine development, although scholars worldwide have carried out extensive research work, to date, no commercial vaccine against Turbot circovirus has been approved for marketing globally. It is worth noting that current research on aquatic vaccines mostly focuses on monovalent vaccines. Such products usually provide immune protection against only a single pathogen. To achieve simultaneous prevention and control of multiple pathogens, multiple and various types of immunization operations are often required. This immunization strategy not only significantly increases the cost input of aquaculture production but also greatly enhances the complexity of production management and the technical threshold. From the analysis of pathogenic microbiological characteristics,E. piscicidais an intracellular parasitic bacterium capable of surviving and multiplying within host cells. Compared with traditional inactivated vaccines, live attenuated vaccines are more conducive to mimicking the natural infection process of pathogens. Through continuous, low-level antigen stimulation, they can more effectively activate the body"s innate and adaptive immune responses, particularly inducing specific cellular immune responses, thereby enhancing the host"s immune defense capability against homologous pathogen invasion. Based on the above theoretical foundation, this study aims to use genetic engineering technology to construct a recombinant vector vaccine capable of simultaneously providing immune protection againstE. piscicidaand TCV.