链球菌病是一种高度传染性的水生细菌性疾病,对罗非鱼构成重大威胁。接种疫苗是众所周知的预防和控制鱼类细菌性疾病的有效措施。在各种免疫方法中,浸泡接种简单,可广泛用于水产养殖。此外,纳米载体递送技术已被报道为提高浸泡疫苗免疫效果的有效解决方案。在这项研究中,表面免疫原性蛋白(Sip)被证明是保守的,并且有可能为无乳链球菌提供交叉免疫保护(S.无乳链球菌)和iniae链球菌(S.iniae)通过多序列比对和Western印迹分析。在此基础上,我们表达并获得了重组蛋白rSip,并将其与功能化碳纳米管(CNT)连接,构建了纳米载体疫苗系统CNT-rSip。浸没免疫后,基于血清特异性抗体水平等方面评价了CNT-rSip对上述两种链球菌感染的免疫效果,非特异性酶活性,细菌攻击后免疫相关基因表达和相对存活百分比(RPS)。结果表明,与对照组相比,CNT-rSip显著提高血清抗体水平(P<0.05),相关的酶活性,包括酸性磷酸酶,碱性磷酸酶,溶菌酶和总抗氧化能力活性,以及免疫后2至4周(WPI)的免疫相关基因的表达水平,所有这些指数都在3wpi达到峰值。此外,在实验过程中,CNT-rSip的上述指标均高于不同范围的rSip组。此外,攻击试验表明,CNT-rSip对无乳链球菌和无乳链球菌感染具有交叉免疫保护作用,RPS分别为75%和72.41%,分别,远远高于其他群体。我们的研究表明,纳米载体浸泡疫苗CNT-rSip可以显着提高罗非鱼的抗体效价,并对无乳链球菌和无乳链球菌感染具有交叉免疫保护作用。
Streptococcosis is a highly contagious aquatic bacterial disease that poses a significant threat to tilapia. Vaccination is a well-known effective measure to prevent and control fish bacterial diseases. Among the various immunization methods, immersion vaccination is simple and can be widely used in aquaculture. Besides, nanocarrier delivery technology has been reported as an effective solution to improve the immune effect of immersion vaccine. In this study, the surface immunogenic protein (Sip) was proved to be conserved and potential to provide cross-immunoprotection for both Streptococcus agalactiae (S. agalactiae) and Streptococcus iniae (S. iniae) by multiple sequences alignment and Western blotting analysis. On this basis, we expressed and obtained the recombinant protein rSip and connected it with functionalized carbon nanotubes (CNT) to construct the nanocarrier vaccine system CNT-rSip. After immersion immunization, the immune effect of CNT-rSip against above two streptococcus infections was evaluated in tilapia based on some aspects including the serum specific antibody level, non-specific enzyme activities, immune-related genes expression and relative percent survival (RPS) after bacteria challenge. The results showed that compared with control group, CNT-rSip significantly (P < 0.05) increased the serum antibody levels, related enzyme activities including acid phosphatase, alkaline phosphatase, lysozyme and total antioxidant capacity activities, as well as the expression levels of immune-related genes from 2 to 4 weeks post immunization (wpi), and all these indexes peaked at 3 wpi. Besides, the above indexes of CNT-rSip were higher than those of rSip group with different extend during the experiment. Furthermore, the challenge test indicated that CNT-rSip provided cross-immunoprotection against S. agalactiae and S. iniae infection with RPS of 75 % and 72.41 %, respectively, which were much higher than those of other groups. Our study indicated that the nanocarrier immersion vaccine CNT-rSip could significantly improve the antibody titer and confer cross-immuneprotection against S. agalactiae and S. iniae infection in tilapia.