南美白对虾肝胰腺蛋白提取方法优化及无水保活环境胁迫对细胞凋亡蛋白的影响

Optimization of Protein Extraction from Hepatopancreas of Litopenaeus vannamei and Effect of Environmental Stress during Waterless Transportation on Apoptosis-Related Proteins

为提高Western Blotting结果的可靠性,以蛋白溶出率和凝胶电泳蛋白条带完整性为指标,比较提取液种类、研磨方式、酶抑制剂种类及其体积分数和提取时间对南美白对虾肝胰腺蛋白提取效果的影响。采用优化后的提取方法获得高质量蛋白样品,并采用Western Blotting法分析无水环境胁迫后南美白对虾肝胰腺组织中细胞凋亡信号通路相关蛋白的表达水平。结果表明:RIPA裂解液作为提取溶剂所得的蛋白溶出率高于水提和磷酸盐缓冲液,电动匀浆和液氮研磨所得蛋白条带更完整,4%蛋白酶磷酸酶混合抑制剂能有效抑制肝胰腺内源酶引起的蛋白降解;采用Western Blotting法分析无水保活期间南美白对虾肝胰腺蛋白,发现低温诱导休眠的同时会引起细胞轻微凋亡,且凋亡水平呈应激时间依赖性增加,环境胁迫解除后有所回调。

In the present study,in order to improve the reliability of Western blotting results,the effects of extraction buffers,grinding methods,inhibitor type and concentration,and extraction time on protein extraction from the hepatopancreas of Litopenaeus vannamei were explored by considering protein dissolution rate and the completeness of protein bands in electrophoretic gels.Next,the expression levels of apoptosis-related proteins in the hepatopancreas of Litopenaeus vannamei were detected with Western blotting.The results showed that the protein dissolution rate was higher using RIPA lysis buffer compared with phosphate buffer saline(PBS)or water as the extraction solvent.Electric homogenization and liquid nitrogen grinding provided more complete protein bands.The degradation of proteins by endogenous proteases was obviously suppressed by addition of 4%protease and phosphatase inhibitor cocktail.In addition,Western blotting results revealed that in addition to inducing Litopenaeus vannamei into dormancy,low temperatures induced slight apoptosis in the hepatopancreas tissue.Furthermore,the apoptosis level increased with waterless duration and this effect was alleviated after removal of environmental stress.