猪笼液蛋白酶消减牛乳蛋白致敏表位的研究

李婷婷; 邱毓; 李欣; 陈红兵

doi:10.13386/j.issn1002-0306.2024040034

猪笼液蛋白酶消减牛乳蛋白致敏表位的研究

Ablation to Allergen-Epitopes of Cow's Milk Proteins by Proteases from Pitcher Fluid of Nepenthes×Mirabilis (Lour.) Druce

摘要

摘要: 目的：利用废弃猪笼液中的蛋白酶来消减牛乳主要致敏原蛋白的致敏性。方法：首先采集了米兰达猪笼草（Nepenthes×Miranda）的猪笼液，经过滤、5 kDa膜超滤、真空冷冻干燥和复溶获得酶活为126.276 U/mL的猪笼液蛋白酶液。然后用此酶对牛乳蛋白进行酶解，通过液相色谱-二级质谱法（LC-MS/MS）鉴定酶解后肽段产物，对比致敏原表位数据库以判断蛋白酶对表位的酶切情况。结果：牛乳主要致敏原蛋白中P1位的K、V、F、R、Y、I、S和L以及P1’位的D、A、V、I、L、F、E、R、Y、N、T、Q、S、W和P的肽键被有效酶解，酶切位点位于相对应的表位内，且位于蛋白表面和内部的表位均有被酶解。其中，来自α-LA的表位E_α-LA1中的K-V以及表位E_α-LA2中的K-V和K-A被酶解频率较高，来自β-LG的被酶解频率高的表位处的肽键为E_β-LG1和E_β-LG2中的L-D、V-Y和Y-V，来自αs1-CN的表位E_αs1-CN1中大部分的R-F和F-F被高频酶解，来自αs2-CN的表位E_αs2-CN1中的L-N、R-Y、K-F和K-T和表位E_αs2-CN2中的K-T、K-L和K-I被酶解频率较高，β-CN中被酶解频率较高的表位为E_β-CN1和E_β-CN2中的L-Q、S-W、D-V、L-T、T-D和E-N。结论：猪笼液蛋白酶水解对牛乳蛋白致敏原蛋白表位表现出不同程度的破坏作用，猪笼液有望成为新型蛋白酶开发的资源，实现农业废弃物再利用。

Abstract: Objective: The aim of this study was to abate the allergenicity of the major allergenic proteins in cow's milk by utilizing the proteases in pitcher fluids of Nepenthes mirabilis (Lour.) Druce. Methods: The pitcher fluid of Nepenthes×Miranda was firstly collected, and the protease solution with the proteolytic activity of 126.276 U/mL was obtained by filtration, ultrafiltration with a 5 kDa membrane, vacuum freeze-drying and re-solubilization. Next, these enzymes were used to digest proteins of cow's milk, and the products were identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Then the peptide products were compared with database of allergen-epitopes, so as to determine the cleavage of the allergen-epitopes. Results: The peptide bonds of K, V, F, R, Y, I, S as well as L in the P1 positions and D, A, V, I, L, F, E, R, Y, N, T, Q, S, W as well as P in the P1' positions of the major allergenic proteins in cow's milk were effectively digested, and the cleavage sites were located in the linear epitopes. What's more, both epitopes located on the surface of the proteins and those located in the interior were digested. Specifically, K-V in epitope E_α-LA1 from α-LA and K-V as well as K-A in epitope E_α-LA2 were cleaved at a high frequency. Peptide bonds at epitopes from β-LG which were cleaved at a high frequency were L-D, V-Y, and Y-V in E_β-LG1 and E_β-LG2. The most of R-F and F-F in epitope E_αs1-CN1 from αs1-CN were cleaved at high frequencies. What's more, L-N, R-Y, K-F as well as K-T in epitope E_αs2-CN1 from αs2-CN and K-T, K-L as well as K-I in epitope E_αs2-CN2 were digested with high frequency. And L-Q, S-W, D-V, L-T, T-D as well as E-N in epitopes E_β-CN1 and E_β-CN2 from β-CN were digested with high frequency. Conclusion: The proteases from pitcher fluid exhibited different destructive effects on the allergen-epitopes of cow's milk proteins. Besides, the pitcher liquid of Nepenthes×Miranda is expected to be the resource for the development of novel proteases and the reuse of agricultural wastes will be achieved.