Guangxi Key Laboratory of Aquatic Genetic Breeding and Healthy Aquaculture, Guangxi Institute of Fisheries, Nanning, PR China.
Guangxi Key Laboratory of Veterinary Biotechnology, Guangxi Veterinary Research Institute, Nanning, PR China.
Fish Shellfish Immunol. 2019 Nov;94:398-406. doi: 10.1016/j.fsi.2019.09.028. Epub 2019 Sep 12.
Crustin is an antimicrobial peptide (AMP) that plays a key role in the innate immunity of crustaceans. This study cloned a new crustin from Pacific white shrimp Litopenaeus vannamei, which we designated as LvCrustinB, using rapid amplification of cDNA ends (RACE). The full-length cDNA of LvCrustinB is 751 bp with an open reading frame (ORF) of 591 bp encoding a peptide of 196 amino acids that includes a putative signal sequence. LvCrustinB is a type II crustin that has a glycine-rich region and a single whey acidic protein domain (WAP) domain. The mRNA transcript of LvCrustinB was detected in all examined tissues and was found to be most abundantly expressed in the epithelium and muscle. The expression of LvCrustinB in hemocytes was significantly upregulated after L. vannamei was challenged with LPS, Vibrio parahaemolyticus, and white spot syndrome virus (WSSV). When LvCrustinB was knocked down with RNAi, the mortality rate of L. vannamei significantly increased after V. parahaemolyticus or WSSV infection. Recombinant LvCrustinB was produced using Pichia pastoris GS115 and was shown to bind to 2 g-positive bacteria (Staphylococcus aureus and Bacillus subtilis) and 2 g-negative bacteria (Escherichia coli and V. parahaemolyticus) via polysaccharides, which included PGN, LTA, and LPS. In vivo, the recombinant LvCrustinB remarkably protected L. vannamei from V. parahaemolyticus infection. These results suggest that LvCrustinB plays an important role in innate immunity and may be potentially utilized as antibacterial agents in shrimp.
抗菌肽 Crustin 在甲壳动物的先天免疫中起着关键作用。本研究使用快速扩增 cDNA 末端（RACE）技术，从凡纳滨对虾（Litopenaeus vannamei）克隆了一种新的 Crustin，我们将其命名为 LvCrustinB。LvCrustinB 的全长 cDNA 为 751bp，开放阅读框（ORF）为 591bp，编码一个 196 个氨基酸的肽，其中包括一个推定的信号序列。LvCrustinB 是一种 II 型 Crustin，具有富含甘氨酸的区域和一个乳清酸性蛋白（WAP）结构域。LvCrustinB 的 mRNA 转录本在所有检测的组织中均有检测到，在表皮和肌肉中表达最为丰富。LvCrustinB 在血细胞中的表达在凡纳滨对虾受到 LPS、副溶血弧菌和白斑综合征病毒（WSSV）刺激后显著上调。当用 RNAi 敲低 LvCrustinB 时，凡纳滨对虾在感染副溶血弧菌或 WSSV 后死亡率显著增加。使用巴斯德毕赤酵母 GS115 生产重组 LvCrustinB，结果表明其通过多糖（包括 PGN、LTA 和 LPS）与 2g 阳性菌（金黄色葡萄球菌和枯草芽孢杆菌）和 2g 阴性菌（大肠杆菌和副溶血弧菌）结合。体内实验表明，重组 LvCrustinB 显著保护凡纳滨对虾免受副溶血弧菌感染。这些结果表明，LvCrustinB 在先天免疫中起着重要作用，可能有望作为虾类的抗菌剂。
