首页|昆虫融合抗冻蛋白提取方法的比较

昆虫融合抗冻蛋白提取方法的比较

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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.
Comparison of methods for extraction of insect fusion antifreeze protein
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.

Antifreeze protein(AFP)Low temperature ethanol methodAmmonium sulfate salting-out methodIsoelectric point methodPurityRelative yield of protein

麻强强、谭婷婷、寇丁月、海瑜涵、邓娅娅、雷喆、吕贵珍、王岩

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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

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

国家自然科学基金国家自然科学基金新疆生产建设兵团科技创新人才项目新疆生产建设兵团科技计划石河子大学创新发展专项国家级大学生创新创业训练计划中国博士后科学基金面上项目

32060125815606142023CB008-182023AB049-4CXFZ2022122024-107590792016M602907

2024

中国生物制品学杂志
中华预防医学会,长春生物制品研究所有限责任公司

中国生物制品学杂志

CSTPCD
影响因子：0.417
ISSN：1004-5503
年,卷(期)：2024.37(5)
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