昆虫融合抗冻蛋白提取方法的比较
Comparison of methods for extraction of insect fusion antifreeze protein
麻强强 1谭婷婷 1寇丁月 1海瑜涵 1邓娅娅 1雷喆 1吕贵珍 1王岩2
作者信息
- 1. 石河子大学医学院,新疆石河子 832000
- 2. 石河子大学医学院,新疆石河子 832000;石河子大学动物科技学院博士后流动站,新疆石河子 832000
- 折叠
摘要
目的 对用低温乙醇法、硫酸铵盐析法及等电点法提取的昆虫融合抗冻蛋白EGFP-OcAFP4浓度和蛋白相对得率进行比较.方法 选取不同终浓度的乙醇(5％～65％)、硫酸铵(0.2～3.0mol/L)及pH(5.6、5.4、5.2、5.0、4.8、4.7、4.6、4.5、4.4)提取重组质粒pET28a-EGFP-OcAFP4诱导表达液破菌上清中的EGFP-OcAFP4,并检测其纯度及蛋白相对得率.结果 乙醇终浓度为30％时提取EGFP-OcAFP4的纯度最高,达86.21％,蛋白相对得率为6.89％;硫酸铵终浓度为2.0 mol/L时提取EGFP-OcAFP4的纯度最高,达42.33％,蛋白相对得率为30.83％;pH为5.0时提取EGFP-OcAFP4的纯度最高,达15.46％,蛋白相对得率为11.97％.结论 低温乙醇法、硫酸铵盐析法和等电点法均可用于EGFP-OcAFP4的提取,但低温乙醇法和硫酸铵盐析法提取蛋白的纯度及相对蛋白得率更高,更适用于EGFP-OcAFP4的提取.
Abstract
Objective To compare the concentration and protein relative yield of insect fusion antifreeze protein EGFP-OcAFP4 extracted by low temperature ethanol method,ammonium sulfate salting-out method and isoelectric point method.Methods By screening different concentrations of ethanol(5％-65％)and ammonium sulfate(0.2-3.0 mol/L)and different the pH values(5.6,5.4,5.2,5.0,4.8,4.7,4.6,4.5,4.4),EGFP-OcAFP4 was extracted from the supernatant of the induced expression solution of recombinant plasmid pET28a-EGFP-OcAFP4,and detected for the purity and protein relative yield.Results When prepared by ethanol method,EGFP-OCAFP4 had the highest purity of 86.21％at the ethanol final concen-tration of 30％,and the relative yield was 6.89％;when it was prepared by ammonium sulfate salting-out,the purity was up to 42.33％with the protein relative yield of 30.83％at the ammonium sulfate final concentration of 2.0 mol/L;the purity of EGFP-OcAFP4 extracted by isoelectric point method with pH of 5.0 was the highest 15.46％,and the relative yield was 11.97％.Conclusion Low temperature ethanol method,ammonium sulfate salting-out and isoelectric point method can all be used for the extraction of EGFP-OcAFP4,but low temperature ethanol method and ammonium sulfate salting-out method have higher extraction purity and protein relative yield,which are more suitable for the extraction of EGFP-OcAFP4.
关键词
抗冻蛋白/低温乙醇法/硫酸铵盐析法/等电点法/纯度/蛋白相对得率Key words
Antifreeze protein(AFP)/Low temperature ethanol method/Ammonium sulfate salting-out method/Isoelectric point method/Purity/Relative yield of protein引用本文复制引用
基金项目
国家自然科学基金(32060125)
国家自然科学基金(81560614)
新疆生产建设兵团科技创新人才项目(2023CB008-18)
新疆生产建设兵团科技计划(2023AB049-4)
石河子大学创新发展专项(CXFZ202212)
国家级大学生创新创业训练计划(2024-10759079)
中国博士后科学基金面上项目(2016M602907)
出版年
2024
NETL
NSTL
万方数据