聚乙二醇修饰β-乳球蛋白羧基对其抗原性和结构的影响

doi:10.7506/spkx1002-6630-20200803-039

食品科学 ›› 2021, Vol. 42 ›› Issue (24): 16-23.doi: 10.7506/spkx1002-6630-20200803-039

• 食品化学 • 上一篇

聚乙二醇修饰β-乳球蛋白羧基对其抗原性和结构的影响

罗舜菁，吉莉，熊绍百，钟俊桢，朱晓明，江辛琳，刘成梅

（1.南昌大学食品科学与技术国家重点实验室，江西南昌 330047；2.江西省辅食营养食品工程技术研究中心，江西赣州 341000）

发布日期:2021-12-30
基金资助:
国家自然科学基金地区科学基金项目（31560435）

Effect of PEGylation of β-Lactoglobulin at Carboxyl Residues on Its Structure and Antigenicity

LUO Shunjing, JI Li, XIONG Shaobai, ZHONG Junzhen, ZHU Xiaoming, JIANG Xinlin, LIU Chengmei

(1. State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang 330047, China;2. Jiangxi Province Engineering Research Center of Fortified Complementary Foods, Ganzhou 341000, China)

Published:2021-12-30

摘要/Abstract

摘要： 采用聚乙二醇（polyethylene glycol，PEG）对β-乳球蛋白（β-lactoglobulin，β-LG）羧基共价修饰，探究PEG修饰及PEG化程度对β-LG抗原性和结构的影响。结果表明：总修饰率为82.91%；使用SP?Sepharose?Fast?Flow阳离子交换柱分离纯化，获得纯度分别为99.66%和98.61%的两种主要修饰产物；十二烷基硫酸钠-聚丙烯酰胺凝胶电泳及基质辅助激光解析电离飞行时间质谱鉴定产物分子质量分别为23.3?kDa和28.6?kDa，为单修饰产物（mono-PEG-β-LG）及两点修饰产物（di-PEG-β-LG）。β-LG、mono-PEG-β-LG和di-PEG-β-LG的间接竞争酶联免疫吸附测定半抑制浓度（IC50）分别为1.90、2.47?μg/mL和10.41?μg/mL；mono-PEG-β-LG和di-PEG-β-LG的IC50分别是β-LG的1.30?倍和5.48?倍，表明提高PEG修饰程度可极显著降低β-LG的抗原性。mono-PEG-β-LG和di-PEG-β-LG的游离巯基含量分别为42.70?μmol/g和37.97?μmol/g，表明PEG化程度增强，遮蔽作用增强。表面疏水性和内源荧光分析表明，mono-PEG-β-LG只发生了三级结构变化，而di-PEG-β-LG二级结构水平同时发生变化，表明PEG修饰后β-LG空间结构的改变可能是导致其构象表位破坏和抗原性下降的因素。PEG分子遮蔽可能是导致修饰产物抗原性降低的另一原因。PEG共价修饰β-LG可以显著降低后者的抗原性，且随PEG化程度增大β-LG抗原性降低幅度增加。

关键词: β-乳球蛋白；聚乙二醇修饰；抗原性；结构变化

Abstract: The effect of PEGylation and PEGylation degree on the antigenicity and structure of β-lactoglobulin were investigated by covalent binding of PEG to the carboxyl group of β-lactoglobulin. The results showed that the total modification rate was 82.91%. The PEGylation products were separated and purified using an SP Sepharose Fast Flow cation exchange column into two major fractions with purity of 99.66% and 98.61%. Using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) the relative molecular weights of the products identified as 23.3 and 28.6 kDa, respectively, indicating that they were mono-PEG-β-LG and di-PEG-β-LG. The half-maximal inhibitory concentration (IC50) values of β-LG, mono-PEG-β-LG and di-PEG-β-LG were 1.90, 2.47 and 10.41 μg/mL, respectively as determined by indirect competitive enzyme-linked immunosorbent assay (IC-ELISA). The IC50 values of mono-PEG-β-LG and di-PEG-β-LG were 1.30 and 5.48 times higher than that of β-LG, respectively, which indicated that the antigenicity of β-LG could be significantly reduced by increasing the PEGylation degree. The free sulfhydryl contents of mono-PEG-β-LG and di-PEG-β-LG were 42.70 and 37.97 μmol/g, respectively, indicating that as the PEGylation degree increased, the masking effect increased. Surface hydrophobicity analysis and intrinsic fluorescence analysis showed changes in tertiary structure only for mono-PEG-β-LG, and in both secondary and tertiary structure for di-PEG-β-LG, indicating that the spatial structure changes of β-LG after PEGylation might be the cause of the destruction of its conformational epitope and the decrease of its antigenicity. The masking of PEG molecules might be another reason for the reduced antigenicity of products. The covalent PEGylation of the carboxyl group of β-LG could significantly reduce its antigenicity, and the antigenicity decreased significantly with the increase of PEGylation degree.

Key words: β-lactoglobulin; polyethylene glycol modification; antigenicity; structure change

中图分类号:

TS201.4

罗舜菁，吉莉，熊绍百，钟俊桢，朱晓明，江辛琳，刘成梅. 聚乙二醇修饰β-乳球蛋白羧基对其抗原性和结构的影响[J]. 食品科学, 2021, 42(24): 16-23.

LUO Shunjing, JI Li, XIONG Shaobai, ZHONG Junzhen, ZHU Xiaoming, JIANG Xinlin, LIU Chengmei. Effect of PEGylation of β-Lactoglobulin at Carboxyl Residues on Its Structure and Antigenicity[J]. FOOD SCIENCE, 2021, 42(24): 16-23.

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聚乙二醇修饰β-乳球蛋白羧基对其抗原性和结构的影响

Effect of PEGylation of β-Lactoglobulin at Carboxyl Residues on Its Structure and Antigenicity

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