College of Fisheries, Henan Normal University, Xinxiang, Henan, 453007, China.
College of Fisheries, Henan Normal University, Xinxiang, Henan, 453007, China.
Fish Shellfish Immunol. 2019 Jul;90:12-19. doi: 10.1016/j.fsi.2019.04.055. Epub 2019 Apr 20.
Grass carp reovirus (GCRV) is the main viral pathogen that endangers grass carp seriously. Application of vaccine has been considered to be the most effective way to prevent virus infection. VP56 is a protein encoded by gene segment 7 of grass carp reovirus, and is predicted to share homology with fiber protein of mammalian reovirus (MRV). In our study, the immunogenicity of VP56 was evaluated by neutralization test. GCRV was incubated with mouse anti-VP56 antibody, and then was injected into grass carp. Results showed that disease progress and death occurrence was hindered in the experimental group compared with the control group. For further study, the recombinant VP56 protein (rVP56) expressed by pET-32a (+) vector was purified, and was used as subunit vaccine to immunize grass carp. After each fish (15 ± 1.5 g) was injected with 30 μg purified rVP56 intraperitoneally, the immune protective efficacy of recombinant VP56 protein was assessed by a series of immune parameters. The population of red blood cells in immunized fish increased significantly after 5 d post injection (dpi), and reached a peak with (2.98 ± 0.17) × 10/ml at 7 dpi (p < 0.05). The numbers of white blood cells peaked with (8.42 ± 1.01) × 10/ml at 7 dpi (p < 0.05). Additionally, the percentage of monocytes and neutrophils rose to a peak with (9.05 ± 0.92)% and (25.93 ± 2.60)% respectively at 5 dpi (p < 0.05 or p < 0.01), whereas lymphocytes reached the highest value of (85.81 ± 2.73) % at 14 dpi (p < 0.01). Serum antibody titer in the vaccinated fish increased significantly and reached a peak at 21 dpi (p < 0.01). The mRNA expression levels of type I interferon (IFN1), major histocompatibility complex class I (MHC I), Toll-like receptor 22 (TLR22), and immunoglobulin M (IgM) were significantly up-regulated in head kidney and spleen (p < 0.05 or p < 0.01). The GCRV challenge test showed that the relative survival rate in immunized group was 71%-75%. Collectively, the results indicated that rVP56 protein can induce immune protection in grass carp, and can be consider as a candidate vaccine against GCRV infection.
草鱼呼肠孤病毒（GCRV）是严重危害草鱼的主要病毒病原体。应用疫苗被认为是预防病毒感染的最有效方法。VP56 是草鱼呼肠孤病毒基因片段 7 编码的一种蛋白质，据预测与哺乳动物呼肠孤病毒（MRV）的纤维蛋白具有同源性。在我们的研究中，通过中和试验评估了 VP56 的免疫原性。将 GCRV 与小鼠抗-VP56 抗体孵育，然后注入草鱼体内。结果表明，实验组的疾病进展和死亡发生率低于对照组。为了进一步研究，用 pET-32a（+）载体表达的重组 VP56 蛋白（rVP56）被纯化，并用作亚单位疫苗免疫草鱼。每尾鱼（15±1.5g）经腹腔注射 30μg 纯化的 rVP56 后，通过一系列免疫参数评估重组 VP56 蛋白的免疫保护效力。免疫后 5 天（dpi），草鱼红细胞数量显著增加，7 dpi 时达到峰值（2.98±0.17）×10/ml（p<0.05）。白细胞数量在 7 dpi 时达到峰值（8.42±1.01）×10/ml（p<0.05）。此外，单核细胞和中性粒细胞的百分比分别在 5 dpi 时上升至峰值（9.05±0.92）%和（25.93±2.60）%（p<0.05 或 p<0.01），而淋巴细胞在 14 dpi 时达到最高值（85.81±2.73）%（p<0.01）。免疫鱼血清抗体滴度显著升高，在 21 dpi 时达到峰值（p<0.01）。头肾和脾脏中 I 型干扰素（IFN1）、主要组织相容性复合体 I（MHC I）、Toll 样受体 22（TLR22）和免疫球蛋白 M（IgM）的 mRNA 表达水平显著上调（p<0.05 或 p<0.01）。草鱼呼肠孤病毒攻毒试验表明，免疫组的相对存活率为 71%-75%。综上所述，结果表明 rVP56 蛋白可诱导草鱼产生免疫保护，可作为草鱼呼肠孤病毒感染的候选疫苗。
