利用單株抗體探討牛乳β-lactoglobulin之結構及特性

Abstract

乳製品中加熱殺菌是必要的過程，而蛋白質之結構會因為加熱殺菌過程中改變，在本研究中，我們利用物理、生物化學及免疫學之方式深入探討蛋白質之變化。利用native-PAGE分析不同品牌之鮮乳及還原乳發現，部分品牌中之乳蛋白β-lactoglobulin (β-LG)結構有90%改變，但α-lactoalbumin (α-LA)結構並未改變。進一步利用Western blot分析加工後之乳製品中之乳蛋白，其結果發現部分結構改變之β-LG會形成多種大分子之結構。利用circular dichroic (CD)分析β-LG經不同時間(5-960秒)及不同溫度(50-95℃)加熱後結構之改變，此加熱條件導致在ellipticity在205 nm觀察其結構由β-sheet轉變成不規則之結構，發現溫度低於70℃加熱達480秒其結構無顯著之改變，但在加熱80-95℃其結構會隨著加熱時間快速且顯著性變化，在15秒即有50%之β-LG結構達到最大改變。此外當β-LG加熱超過80℃結構改變後會完全喪失與retinol結合及其特性。因此檢測乳製品中蛋白質結構之改變可達監控乳製品品質之目的。然而現今並無利用免疫化學方式測定鮮乳或還原乳中蛋白質結構之改變。本研究中目的在於製作可區分還原乳及生乳之單株抗體，因此我們將還原乳直接免疫小鼠製作單株抗體。在900株hybridoma中發現有四株能專一性辯識還原乳，且此單株抗體能直接偵測生乳中摻雜還原乳，其靈敏度能偵測生乳中僅添加5%還原乳。有趣的是利用Western blot分析此四株單株抗體皆辨識β-LG及與β-LG結合之大分子結構。其中一株專一性抗體辨認區位於β-LG D strand (第66-76胺基酸)，此外此結構上之第69胺基酸Lys與retinol及palmitic acid結合有關，當此區加熱結構改變後即會被此抗體辨識，但結合retinol及palmitic acid之能力會完全喪失。綜合上述結果β-LG為鮮乳中重要之溫度指標。為了評估乳製品之品質，我們另外製作了一株單株抗體僅辨識native β-LG，當β-LG經加熱結構改變後即不會被此抗體辨識。最後我們更證明出當β-LG受熱結構改變後會喪失刺激B-lymphocytes增生之功能，此外我們發現刺激B-lymphocytes增生之功能是經由receptor-mediate，亦證明出此receptor為IgM。

Heating process is necessary for milk manufacture. However, the structures and properties of some protein existed in milk may change during this procedure. In this study, we utilized physical, biochemical, and immunological methods to study such structural changes. Using a native-polyacrylamide gel electrophoresis (PAGE), 90% of β-lactoglobulin (β-LG) was denatured in some brands of the processed and dry milk, but not α-lactoalbumin (LA). By Western blot, a part of the denatured β-LG formed amounts of multimeric ones in the processed product. The thermal denaturation processes of β-LG and LA were monitored at various temperatures (50-95 °C) and duration (5-960 sec) via a circular dichroic analysis. The heating conditions might influence the ellipticity at 205 nm reflecting the conformational changes from β-structure to disordered structure. There were no significant conformational changes of β-LG at the temperature below 70 °C for as long as 480 sec. However, pronounced and rapid changes occurred between 80-95 °C with a time-dependent fashion. Fifty percent of the maximal changes achieved within 15 sec. The heated β-LG above 80 °C was almost completely lost its binding property with retinol. Nevertheless, there is no immunochemical method that can be employed for the detection of protein structural change in processed or dry milk. This study aims to develop a sensitive monoclonal antibody (mAb) that could distinguish the dry milk from freshly prepared raw milk. Therefore, commercially prepared dry milk was used to immunize mice for production of mAb. From 900 hybridomas, 4 clones were found to be specific to dry milk. The specific mAbs could detect the dry milk spiked into the raw milk as low as 5% in concentration (v/v). Most interestingly, the 4 mAbs were all against β-LG and LG-milk protein conjugates in western blot. Furthermore, the epitope was characterized to be located within the D strand of β-LG (residues 66-76). In this region, lysine (residue 69) was associated with β-LG binding ability of retinol and palmitic acid. After heating process, it completely lost its binding ability. According to these results, β-LG could be regarded as a thermal marker in processed milk. To completely evaluate the quality of dairy products, we also produced a mAb specific against native β-LG. Eventually, we speculated that the conformational changes of β-LG during the heating process leads to the loss of its ability in stimulating B-lymphocytes proliferation.