摘要
牛乳是主要的食物过敏原，避免摄入牛乳过敏原成分是预防牛乳过敏有效途径。目前，我国国内尚未对食品进行过敏原标签化管理，急需建立牛乳过敏原检测的相关标准和方法。因此，开展牛乳过敏原成分检测的研究十分必要。
β-乳球蛋白（β-lactoglobulin，βLG）是牛乳的主要过敏原，被认为是检测食品中牛乳过敏原的特异性标志蛋白。同时，目前用于消除βLG致敏性的方法都有一定的局限性，寻求一种有效降低βLG的致敏性并保持其功能的有效手段对开展低致敏乳制品的研究具有十分重要的意义。
本研究以牛乳βLG作为研究对象，基于βLG的免疫反应性建立起检测食物中βLG的方法。同时将茶多酚的主要活性成分没食子儿茶素没食子酸酯（epigallocatechingallate，EGCG）和没食子儿茶素（epigallocatechin，EGC）与βLG结合，分析茶多酚的主要活性成分如何与βLG结合及其对蛋白构象产生的影响，评价βLG结合茶多酚的主要活性成分后蛋白免疫反应性的变化，探讨βLG结合茶多酚活性成分后免疫反应性发生改变的机理。主要研究结果如下：
（1）制备获得了八株抗β-乳球蛋白的单克隆抗体。这八株抗体纯度较高、特异性强、效价高、没有交叉反应。
（2）利用合成的4个βLG主要IgG抗原表位的多肽片段，通过间接法ELISA和免疫点杂交检测所获得的八株单抗与4个βLG主要IgG抗原识别的情况。结果表明，所制备单克隆抗体1G5识别的抗原表位为51Glu-64Asp；1H8识别的抗原表位为67Arg-88Asn；3D11和4C3均识别的抗原表位为与129Asp-144Pro。
（3）通过抗体筛选和配对，将已知抗原表位的单克隆抗体1G5和1H8作为配对抗体，建立了四种检测方法（双抗体夹心ELISA法，时间分辨免疫荧光法、免疫胶体金法和免疫印迹法）系统性检测食物中的牛乳βLG成分。双抗体夹心ELISA法检出限约为1.6ng mL~(-1)；时间分辨免疫荧光法灵敏度约为0.2ng mL~(-1)；免疫胶体金法检出低限约为0.2ng mL~(-1)，其比传统的ELISA方法相比更简便和快捷。
（4）通过荧光滴定法实验推断EGCG淬灭βLG内部荧光过程是一个静态淬灭过程，EGC淬灭βLG内部荧光过程是动态淬灭和静态淬灭同时存在的过程。EGCG和EGC与βLG相互结合的过程都是自发过程，EGCG与βLG结合可能是以疏水作用为主，EGC与βLG结合可能是以氢键和范德华力为主。
（5）通过多光谱技术对蛋白的构象变化进行分析可知：EGCG和EGC可结合βLG，覆盖在蛋白的表面并对βLG的构象产生影响，但影响有限，蛋白的α-螺旋结构和β-折叠结构有所增加，蛋白分子的球形结构保持完整，EGCG和EGC使蛋白更为紧凑，对蛋白的整体构象并未产生破坏。同时，蛋白结合EGCG和EGC后，蛋白与IgG和IgE的结合能力有所下降。推断βLG结合EGCG和EGC后免疫反应性降低主要不是由蛋白构象改变造成的，EGCG或EGC可能直接结合在抗原表位上或抗原表位附近，从而使抗体难以识别结合到抗原表位上，导致抗原和抗体结合时在空间上存在位阻。EGCG结合βLG后对蛋白构象和免疫反应性的影响比EGC大。
（6）βLG预热处理后与EGCG结合，其结合能力增强，蛋白免疫反应性的降低程度大于单一的热处理蛋白。
总之，本研究建立起检测牛乳主要过敏原βLG的系列方法，填补国内的检测牛乳过敏原的空白。同时通过探讨βLG结合茶多酚主要活性成分后免疫反应性发生改变的机理，将为低致敏乳制品的开发提供新的思路和理论支持。
Allergy to cow’s milk proteins is a common food allergy. Specific avoidance of cow milkis the only way to prevent milk allergies. However, there is yet no domestic food-labeling lawfor food allergens. It is necessary to establish methods for the detection of milk contaminationin food products.
Bovine β-lactoglobulin (βLG) is a major allergen of cow milk and considered to be aspecific milk allergen marker in food products. The current widely used methods to diminishallergenicity of βLG all have limitations. It is crucial to develop new methods that wouldreduce the allergenicity without destroying functional properties of βLG.
Our research focused on βLG. A series of detection methods for βLG in food productswere established based on the immunoreactivity of βLG. We prepared βLG-catechincomplexes by binding the protein with epigallocatechingallate (EGCG) and epigallocatechin(EGC), respectively. We explored how EGCG and EGC bound to βLG and the structurechanges of βLG before and after these catechins binding. Then we estimatedimmunoreactivity of these complexes. The present research aimed to perform a mechanisticstudy concerning immunoreactivity changes of βLG before and after catechins binding. Thefollowing six aspects of research are listed below:
（1）Eght monoclonal antibodies (mAbs) against βLG allergen were prepared andidentificated. The results demonstrated that these mAbs were pure, specific without anycross-reactivity toward other food allergen sources.
（2）Four major IgG epitopes of βLG were synthesized and probed by the mAbs throughindirect ELISA and immuno dot-blotting. The results showed that the epitopes recognized by1G5was located in the region51Glu-64Asp; for1H8,67Arg-88Asn; for3D11and4C3,129Asp-144Pro.
（3）Two mAbs (1H8and1G5) with identified epitope regions were paired anddeveloped into detection methods of βLG allergen. The detection methods included doubleantibody sandwich ELISA (S-ELISA), time-resolved fluoroimmunoassay (TRFIA), goldimmunochromatography assay (GICA) and western blotting (WB). The detection limit ofS-ELISA, TRFIA and GICA were1.6ng mL~(-1),0.2ng mL~(-1)and0.2ng mL~(-1), respectively.GICA method described here is much simpler and faster than an ELISA assay.
（4）The binding interactions between EGCG and EGC which βLG were thoroughlystudied by fluorescence quenching. It indicated that the nature of EGCG quenching was static,while the nature of EGC quenching involved a combined quenching (static and dynamic) process. The thermodynamic parameters of the interaction between EGCG and EGC withβLG indicated that the interactions process was spontaneous. The results suggested thathydrophobic forces play a major role in the binding between EGCG and βLG, Van der Waalsinteractions and hydrogen bonds play major roles between EGC and βLG.
（5）The results of multi-spectroscopic experiments revealed that βLG bound by EGCGand EGC, which resulted in slight change of native conformation of βLG without apparentdisruption. It was found that the α-helical and β-sheet content of the protein were slightlyincreased. The conformation of βLG was little more compact after EGCG and EGC binding.The reduction of the binding affinity of βLG-catechins complexes with antibodies may due tothe shielding action of the βLG epitopes induced by the attachment of catechins to βLG.EGCG and EGC in βLG-catechins complex may be attached directly to the epitope(s) orregion(s) in the proximity, thereby preventing anti-βLG antibodies from accessing theantigens. The effect of EGCG was greater than EGC.
（6）Thermal treatment on protein would increases binding affinity of EGCG with βLG.EGCG was attached on the preheat βLG，which can reduced the binding activities betweenantigen and antibodies greater than EGCG bingding βLG without preheat.
In general, a series of detection methods for βLG in food products were established,which filled up domestic blank. At the same time, we examined the mechanismimmuoreativity changes of βLG before and after tea polypheols binding. We hope thatour research will provide new alternative and theoretic supports for development ofhypoallergenic milk products.
引文
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