Extraction process of intestinal lipid droplets and lipid droplet-related proteins from tilapia
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摘要: 动植物中的脂质以脂滴 (Lipid droplet, LD) 的形式存在。掌握脂滴及脂滴相关蛋白 (Lipid droplet related protein, LDRP) 的提纯方法，对解析鱼类脂质变化及影响机制，寻求适宜的调控途径至关重要。以罗非鱼 (Oreochromis niloticus) 的肠系脂肪组织为原料，利用离心、重悬及增溶等处理手段分离纯化脂滴及LDRP，并结合SDS-PAGE和质量浓度变化分析提纯效果。结果表明，肠系脂肪组织经3次离心和3次重悬可有效去除杂质，获得较为纯净的脂滴。虽然溶解剂增溶提取的LDRP蛋白浓度较高，但电泳条带存在明显拖尾现象，说明脂质脱除效果不佳，影响了LDRP提取纯度。脱脂增溶需经4组有机溶剂联合处理才可获得纯度高、复溶效果好的LDRP提取样品。离心、干燥方式及脂滴冷冻对LDRP增溶影响差异不显著。Abstract: Lipids in animals and plants exist in the form of lipid droplet (LD). It is very important to master the purification methods of lipid droplet and lipid droplet related protein (LDRP) for understanding the lipid changes and influence mechanisms of fish, and seeking appropriate regulatory approaches. In this paper, the lipid droplet and LDRP were separated and purified through centrifugation, resuspension and solubilization by using Oreochromis niloticus intestinal tissues as raw material. The results show that the intestinal adipose tissues were effectively removed by three times of centrifugation and three times of resuspension to obtain the pure lipid droplet. Although the concentration of LDRP protein extracted by solubilization was higher, the electrophoretic bands showed an obvious trailing phenomenon, indicating that the effect of lipid removal was poor, which affected the purity of LDRP extraction. Delipidation and solubilization need to be combined with four groups of organic solvents to obtain the LDRP extracted samples with high purity and good re-solubilization effects. The effects of centrifugation drying method and lipid droplet freezing on LDRP solubilization were not significantly different.
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Key words:
- Oreochromis niloticus /
- Lipid droplet /
- Lipid droplet-related protein /
- Extraction
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图 1 肠系脂肪组织脂滴提取过程
Figure 1. Extraction process of lipid droplet in intestinal adipose tissue
图 2 肠系脂肪组织学观察
Figure 2. Histological observation of tilapia's mesenteric adipose tissue
图 6 溶解剂增溶和脱脂增溶的LDRP质量浓度
Figure 6. LDRP's mass concentration of solubilization by solubiliser and delipidation and solubilization
图 8 离心和干燥方式对脱脂增溶的影响
Figure 8. Effects of centrifuge and drying method on delipidation and solubilization
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