摘要
为探究不同共价修饰位点对β-乳球蛋白(β-lactoglobulin,β-LG)抗原性影响,采用聚乙二醇(polyethylene glycol,PEG)定点修饰技术对β-LG第121位半胱氨酸(Cys121)游离巯基进行修饰。通过单因素试验优化,得到最佳修饰条件:β-LG与mPEG-MAL物质的量比1∶30,在0.1 mol/L,pH 7.0的磷酸钠缓冲液(含3倍物质的量的TCEP,25%乙醇及2 mmol/L EDTA)中4℃反应24 h。使用优化条件制备产物修饰率达64.1%,分离纯化后产物纯度达94%。游离巯基含量测定结果显示修饰后β-LG游离巯基含量由52.3 μmol/g降为0.3μmol/g。经验证mPEG-MAL与β-LG Cys121游离巯基发生特异性结合。经巯基定点修饰后,β-LG-MAL较天然β-LG抗原性显著提升25.9%,巯基修饰与前期不同位点修饰抗原性结果相反,定点修饰能有效探究不同位点共价修饰对蛋白抗原性的影响。
In order to investigate the effect of different covalent modification sites on the antigenicity of β-lactoglobulin(β-LG), β-LG was modified by using cysteine-specific PEGylation at the thiol group of Cys121. Using one-factor-at-a-time method, the optimal modification conditions were determined as follows: β-LG and m PEG-MAL(molar ratio, 1:30) were dissolved together in 0.1 mol/L phosphate buffer containing 0.3 mol/L tris-(2-carboxyethyl) phosphine(TCEP), 25%(V/V) ethanol and 2 mmol/L ethylenediaminetetraacetic acid(EDTA) at pH 7.0 and incubated at 4 ℃ for 24 h, yielding a maximum modification efficiency of 64.1%. The resulting product was purified to a purity of 94%. The free sulfhydryl content of β-LG decreased from 52.3 to 0.3 μmol/g after the site-specific PEGylation. It was demonstrated that m PEG-MAL was specifically conjugated with β-LG at the thiol group of Cys121. The antigenicity of β-LG increased by 25.9% after the cysteine-specific PEGylation, contrary to results previously obtained by modification at other amino acid positions. Accordingly, site-specific PEGylation can allow effective regulation of β-LG antigenicity.
作者
刘成梅
江辛琳
李冬梅
周磊
钟俊桢
吉莉
罗舜菁
LIU Chengmei;JIANG Xinlin;LI Dongmei;ZHOU Lei;ZHONG Junzhen;JI Li;LUO Shunjing(State Key Laboratory of Food Science and Technology,Nanchang University,Nanchang 330047,China;Jiangxi Danxia Biotechnology Co.Ltd.,Yingtan 335200,China)
出处
《食品科学》
EI
CAS
CSCD
北大核心
2021年第2期1-7,共7页
Food Science
基金
国家自然科学基金地区科学基金项目(31560435)。
关键词
Β-乳球蛋白
聚乙二醇
共价修饰
定点修饰
抗原性
β-lactoglobulin
polyethylene glycol
covalent modification
site-specific PEGylation
antigenicity
