Chen Yu-Yuan, Chen Jiann-Chu, Kuo Yi-Hsuan, Lin Yong-Chin, Chang Yu-Hsuan, Gong Hong-Yi, Huang Chien-Lun
Department of Aquaculture, College of Life Sciences, Center of the Excellence for the Oceans, National Taiwan Ocean University, Keelung, 20224, Taiwan.
Department of Aquaculture, College of Life Sciences, Center of the Excellence for the Oceans, National Taiwan Ocean University, Keelung, 20224, Taiwan.
Dev Comp Immunol. 2016 Feb;55:144-51. doi: 10.1016/j.dci.2015.10.023. Epub 2015 Oct 30.
Lipopolysaccharide and β-1,3-glucan-binding protein (LGBP), important pattern recognition proteins (PRPs), recognize lipopolysaccharide (LPS) and β-1,3-glucan (βG), known as pathogen-associated molecular patterns (PAMPs), and subsequently trigger innate immunity. Several seaweed polysaccharides and seaweed extracts increase immune parameters and resistance to pathogens. Here, we constructed the expression vector pET28b-LvLGBP and transferred it into Escherichia coli BL21 (DE3) for protein expression and to produce the recombinant protein LGBP (rLvLGBP) in white shrimp Litopenaeus vannamei. We examined the binding of rLvLGBP with seaweed-derived polysaccharides including alginate, carrageenan, fucoidan, laminarin, Gracilaria tenuistipitata extract (GTE), and Sargassum duplicatum extract (SDE), and examined the phenoloxidase activity of shrimp haemocytes incubated with a mixture of rLvLGBP and each polysaccharide. We also examined the binding of rLvLGBP with LPS and βG, and the phenoloxidase activity of shrimp haemocytes incubated with a mixture of rLvLGBP and LPS (rLvLGBP-LPS) or a mixture of rLvLGBP and βG (rLvLGBP-βG). An ELISA binding assay indicated that rLvLGBP binds to LPS, βG, alginate, carrageenan, fucoidan, laminarin, GTE, and SDE with dissociation constants of 0.1138-0.1770 μM. Furthermore, our results also indicated that the phenoloxidase activity of shrimp haemocytes incubated with a mixture of rLvLGBP and LPS, βG, alginate, carrageenan, fucoidan, laminarin, GTE, and SDE significantly increased by 328%, 172%, 200%, 213%, 197%, 194%, 191%, and 197%, respectively compared to controls (cacodylate buffer). We conclude that LvLGBP functions as a PRP, recognizes and binds to LPS, βG, alginate, carrageenan, fucoidan, laminarin, GTE, and SDE, and subsequently leads to activating innate immunity in shrimp.
脂多糖和β-1,3-葡聚糖结合蛋白（LGBP）是重要的模式识别蛋白（PRP），可识别脂多糖（LPS）和β-1,3-葡聚糖（βG），它们被称为病原体相关分子模式（PAMP），随后触发先天免疫。几种海藻多糖和海藻提取物可提高免疫参数并增强对病原体的抵抗力。在此，我们构建了表达载体pET28b-LvLGBP，并将其转入大肠杆菌BL21（DE3）中进行蛋白表达，以在凡纳滨对虾中产生重组蛋白LGBP（rLvLGBP）。我们检测了rLvLGBP与海藻衍生多糖（包括藻酸盐、角叉菜胶、岩藻依聚糖、海带多糖、细基江蓠提取物（GTE）和复瓦状马尾藻提取物（SDE））的结合情况，并检测了与rLvLGBP和每种多糖混合物孵育的对虾血细胞的酚氧化酶活性。我们还检测了rLvLGBP与LPS和βG的结合情况，以及与rLvLGBP和LPS混合物（rLvLGBP-LPS）或rLvLGBP和βG混合物（rLvLGBP-βG）孵育的对虾血细胞的酚氧化酶活性。酶联免疫吸附测定结合试验表明，rLvLGBP与LPS、βG、藻酸盐、角叉菜胶、岩藻依聚糖、海带多糖、GTE和SDE结合，解离常数为0.1138 - 0.1770 μM。此外，我们的结果还表明，与对照（二甲胂酸盐缓冲液）相比，与rLvLGBP和LPS、βG、藻酸盐、角叉菜胶、岩藻依聚糖、海带多糖、GTE和SDE混合物孵育的对虾血细胞的酚氧化酶活性分别显著提高了328%、172%、200%、213%、197%、194%、191%和197%。我们得出结论，LvLGBP作为一种PRP发挥作用，识别并结合LPS、βG、藻酸盐、角叉菜胶、岩藻依聚糖、海带多糖、GTE和SDE，随后导致对虾先天免疫的激活。
