PLGA-LrrG微球在罗非鱼组织细胞分布及内化机制研究

Continual outbreaks of streptococcal diseases in tilapia culture have severely affected the sustainable development of the Chinese tilapia industry in recent years. The prevention and control of streptococcosis has become a major focus and challenge in the tilapia industry. Streptococcus agalactiae is the major causes of streptococcosis in tilapia. S. agalactiae is a gram-positive bacterium with spherical or oval morphology and paired or short-chained arrangement.. The Lancefield serotype classification system, in which streptococci are divided into ten serotypes (Ia, Ib, and II–IX) based on antigenic differences of capsular polysaccharides, classifies S. agalactiae as Group B streptococcus. S. agalactiae, which induces streptococcosis, includes three serotypes, including Ia, Ib, and III. An investigation of isolated S.agalactiae showed high rates of serotypes Ia, Ib, II and III in both humans and fish.. Vaccination is considered the most effective means of preventing and controlling streptococcosis. However, there is a dearth of effective cross-protective vaccines for different S. agalactiae serotypes. As the multiple functional genes involved in capsular polysaccharide synthesis had different structures, they eventually formed capsular antigens with strict specificity.. Leucine-rich repeat protein (LrrG protein) is one of the most conserved surface proteins of S. agalactiae serotypes and exhibits an abundance of the leucine-rich repeat (LRR) protein structural motifs. For this reason, LrrG has been considered an ideal candidate target for vaccine antigen and immunoassay development.. PLGA is a lactic acidglycolic acid copolymer formed by the irregular polymerization of lactic acid and glycolic acid. It is non-toxic and non-stimulating, biodegradable, biocompatible, and it exhibits slow-release properties, for which reasons it was approved by the U.S. Food and Drug Administration as an alternative to biological tissues and as a drug carrier. PLGA microspheres have several advantages that make them useful drug carriers. They can encapsulate vaccines, peptides, proteins, and small-molecule drugs and have broader routes of administration, require fewer administrations and lower doses, foster longer drug action to several months, and foster better control of drug release than other methods. They can also improve drug stability, reduce toxicity and stimulation, and maintain protein immunogenicity. This makes PLGA microspheres attractive carriers in vaccine applications. In large-scale industrial fisheries, a number of unique factors are important to evaluating the efficacy of immunization. Oral immunization involves less damage to the fish, less labor, and less time than other methods and is thus thought to be the best fit for aquatic animal immunization. Many studies have indicated that PLGA application technology, which involves an adjuvant encapsulating immunogens in oral vaccine preparations, is relatively safe and efficient. In the previous, we showed the efficacy of LrrG-PLGA application as an oral vaccine in the prevention and control of streptococcosis and its convenience of administration and ability to improve treatment efficacy. The results showed that immunized tilapias were resistant to S. agalactiae infection, with an effective relative immune protection rate.. The present study builds on our previous reports, which intend to further evaluate the distribution and internalized mechanism of PLGA-LrrG particles in tilapia tissue cells. Based on fluorescence labeling, RT-PCR and subcellular ultramicroscopic technique, the characteristics of cell internalization, subcellular localization and the spatiotemporal expressing information of antigen submission immune-related genes in different tissues will be investigated, which then will be analyzed comprehensively by multivariate statistical analysis models. The results will provide a foundation for the development of fish PLGA effective oral vaccines.

无乳链球菌病防治无论从罗非鱼产业发展还是人畜共患病的角度考虑，都急需有所突破。本项目前期以无乳链球菌跨血清保守型表面蛋白LrrG为抗原，利用纳米多聚物PLGA为传递载体免疫罗非鱼，提高了蛋白抗原口服吸收率，并降低了其易降解的风险，在鱼体内产生了有效的免疫刺激。为进一步探讨PLGA-LrrG微球在罗非鱼组织细胞的内化机制，本项目拟通过荧光标记实时定位、重要抗原递呈免疫相关基因的时空表达及亚细胞超显微技术等手段，获取免疫后不同时间点微球在罗非鱼各组织细胞内分布及内化特征、亚细胞定位特点及实时免疫相关基因的表达信息等参数，将其和微球理化特性等参数转换设置成多元变量，并利用多元统计技术R-Vegan等，综合分析PLGA-LrrG微球在罗非鱼组织细胞中的分布及内化机制。旨在为后期LrrG有效抗原定量和鱼类PLGA口服疫苗的研发提供科学数据。

无乳链球菌病防治无论从罗非鱼产业发展还是人畜共患病的角度考虑，都急需有所突破。本项目在前期基础上，开展的主要研究内容为：完成了基于密码子优化策略的无乳链球菌表面蛋白LrrG的原核表达、纯化及免疫原性研究；完成了PLGA-LrrG荧光双标微球的制备、不同组织细胞和亚细胞区域分布、时空定位和免疫基因表达研究；完成无乳链球菌LrrG同类表面蛋白Sip蛋白的优化、表达及免疫原性研究；完成了无乳链球菌破碎纳米疫苗在不同规格罗非鱼幼鱼不同组织的抗原分布研究，及其免疫效果和免疫基因表达情况研究；完成了基于无乳链球菌表面蛋白sip的乳酸球菌载体疫苗的免疫效果研究。结果表明，优化后的LrrG，表达量显著提高，且仍然具有较好的免疫原性；利用类似方法优化的SIP，也可有效提高其原核表达量，同时优化表达的Sip蛋白仍具有免疫原性；体内分布结果表明，表明PLGA-LrrG微球通过注射和口服微球15min时，微球就能到达罗非鱼的脾、头肾、肝、肠、鳃、脑中，直到72h时，罗非鱼的脾、头肾、肝、肠、鳃、脑还能观察到少量存留的微球。亚细胞定位结果显示其主要分布于细胞质中。菌体细胞抗原被粉碎成微球至纳米颗粒，在不同大小规格的罗非鱼各组织中发现抗原摄取阳性信号，主要分布在鳃、口咽、肾、肝、脾、肠道和鱼皮。1g罗非鱼的IgM、IgT、CD8+和C3的相对峰值表达量显著高于0.1 g和0.5 g组。IgT的差异表达最为显著，C3的峰值表达保持时间最长。0.5 g组和1g组在接种后第二周出现抗体水平峰值，均显著高于0.1g罗非鱼，1g罗非鱼的抗体水平持续时间高于其他两种规格组。接种后3个月，3种不同规格罗非鱼的RPS峰值均高于60%，1g罗非鱼的有效保护期均大于0.1 g和0.5 g组。以上结果为探讨鱼类纳米微球疫苗的特性及功能机制奠定了基础，也为罗非鱼无乳链球菌病粘膜疫苗的开发提供了策略。