Reduction of Allergenicity of Milk Protein by Conjugation with Acidic Oligosaccharides

为了降低牛β-乳球蛋白-海藻酸低聚糖(β-LG-ALGO)的致敏性，改善β-LG的功能性质，通过美拉德反应制备了β-LG-ALGO偶联物。β-LG与ALGO的摩尔比为1：6，等电点为&lt；4.6，低于β-LG的等电点。β-LG的糖结合位点分别为60Lys、77Lys、100Lys、138Lys和141Lys。圆二色谱表明，β-LG与ALGO共轭后，二级结构基本保持不变。荧光研究表明，结合物中Trp周围的构象没有改变，表面覆盖着糖链。用单抗进行结构分析表明，结合物中15Val29Ile和8Lys-19-Trp的构象发生了变化，而天然结构保持在125Thr-135Lys附近。通过与ALGO的偶联，β-LG具有较高的热稳定性和较强的乳化能力。β-LG-ALGO结合物免疫BALB/c、C57BL/6和C3H/He小鼠后，抗β-LG抗体反应明显降低。我们测定了β-LG的B和T细胞表位以及在这些小鼠中识别的结合物，发现β-LG-ALGO结合物的线性表位谱与β-LG相似，但每个表位的免疫应答都显著降低。ALGO对表位的掩蔽被认为是结合物中β-LG免疫原性降低的原因。

Bovine β-lactoglobulin-alginic acid oligosaccharide (β-LG-ALGO) conjugate was prepared by the Maillard reaction to reduce the allergenicity and improve the functional properties of β-LG. The molar ratio of β-LG to ALGO in the conjugates was 1 : 6. The isoelectric point of the conjugate was <4.6, which is lower than that of β-LG. Carbohydrate binding sites in β-LG were identified to be 60Lys, 77Lys, lOOLys, 138Lys and141Lys. CD spectra indicated that secondary structure of β-LG was almost maintained after conjugation with ALGO. Fluorescence studies suggested that the conformation around Trp had not changed in the conjugate and that the surface of the conjugate was covered with saccharide chain. Structural analyzes with monoclonal antibodies indicated that the conformation around 15Val29Ile and 8Lys-19-Trp in the conjugate had changed, while native structure was maintained around 125Thr-135Lys. By conjugation with ALGO, β-LG was endowed with high heat stability and improved emulsifying ability. The antiβ-LG antibody response was markedly reduced after immunization with the β-LG- ALGO conjugates in BALB/c, C57BL/6 and C3H/He mice. We determined the B and T cell epitopes of β-LG and the conjugate recognized in these mice and found that the linear epitope profiles of the β-LG- ALGO conjugate were similar to those of β-LG, while the immune response for each epitope was dramatically reduced. Masking of epitopes by ALGO was considered to be responsible for the decreased immunogenicity of the β-LG in the conjugate.