Department of Aquatic Animal Medicine, College of Fisheries, Huazhong Agricultural University, Wuhan, Hubei, 430070, China; Guangdong Provincial Water Environment and Aquatic Products Security Engineering Technology Research Center, Guangzhou Key Laboratory of Aquatic Animal Diseases and Waterfowl Breeding, Guangdong Provincial Key Laboratory of Waterfowl Healthy Breeding, College of Animal Sciences and Technology, Zhongkai University of Agriculture and Engineering, Guangzhou, Guangdong, 510225, China.
Faculty of Arts, University of British Columbia, Vancouver, British Columbia, V6T1W9, Canada.
Fish Shellfish Immunol. 2018 Sep;80:534-539. doi: 10.1016/j.fsi.2018.06.024. Epub 2018 Jun 12.
Red-spotted grouper nervous necrosis virus (RGNNV) is one of the most important viruses which mainly infects the larva of marine and freshwater fish with high mortality and affects the fishery industry worldwide. Currently, there are no effective vaccines available for the fish larva infected with NNV. Immunoglobulin yolk (IgY) origin of oviparous animals is passed from the blood serum and concentrated in the egg yolk. With the advantages of high yield, cost-effectiveness, and high stability, IgY can be widely used in passive immunization, especially in young animals in which adaptive immunity is not fully developed. In this study, we have cloned and expressed the recombinant capsid protein of RGNNV in Escherichia coli and used as an immunogen for generating specific anti-RGNNV IgY antibody in laying hens. Water-soluble fractions (WSF) of the specific IgY were isolated from egg yolk and purified by two-step precipitation with saturated ammonium sulfate salting. By Enzyme linked immunosorbent assay (ELISA), the titer of the IgY reached a peak at the 6th week post of immunization and had a strong stability at a wide range of temperature, pH, and pepsin enzyme digestion. The purified IgY was competent to neutralize and completely inhibited the RGNNV replication in the grouper fin cell line (GF-1), indicating that it was highly specific and effectively recognized RGNNV. The results will pave a new way for the prevention of RGNNV infection.
红鳍东方鲀神经坏死病毒（RGNNV）是一种重要的鱼类病毒，主要感染海水和淡水鱼类的幼鱼，死亡率高，对全球渔业造成影响。目前，尚无针对感染神经坏死病毒的鱼类幼鱼的有效疫苗。卵生动物的免疫球蛋白卵黄（IgY）来源于血清，并在卵黄中浓缩。IgY 产量高、成本效益高、稳定性好，可广泛应用于被动免疫，尤其适用于适应性免疫尚未完全发育的幼小动物。本研究在大肠杆菌中克隆并表达了 RGNNV 的重组衣壳蛋白，并将其作为免疫原，在产蛋鸡中产生针对 RGNNV 的特异性 IgY 抗体。从卵黄中分离出水溶性部分（WSF），并用饱和硫酸铵两步沉淀法进行纯化。通过酶联免疫吸附试验（ELISA），免疫后第 6 周 IgY 的效价达到峰值，在较宽的温度、pH 和胃蛋白酶消化范围内具有很强的稳定性。纯化的 IgY 能够中和并完全抑制东方鲀鳍细胞系（GF-1）中的 RGNNV 复制，表明其具有高度特异性和对 RGNNV 的有效识别能力。该结果为 RGNNV 感染的预防开辟了新途径。
