1 : 109 表 1 本文实验所用的水产品 Tab. 1 Aquatic products used in this study Mud crab Scylla serrata Chinese mitten crab Eriocheir sinensis Japanese blu

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1 34 1 Vol. 34, No ACTA HYDROBIOLOGICA SINICA Jan., DOI: /SP.J 刘光明梁银龙翁凌苏文金黄园园曹敏杰 (,, ) :,, 38 kd 38 kd pi 4.5, Pen a 1,,, Protein A Sepharose, 经倍稀释后仍能与抗原进行反应, : ; ; ; 中图分类号 : TQ93 文献标识码 : A 文章编号 : (2010) ,, [1], [2], [3], [4 6], kd,,, [7] [8] kd, (Scylla serrata),,,,,,,,,,,,,,, ( 1) 20 2 kg/,,,, 11 ( ) ( 1 11) 5, 11, ; 5 SDS-PAGE Fermentas ; IgG Pierce ; IgE KPL ; New England Biolab 收稿日期 : , 修订日期 : 基金项目 : ( ); (2008J0067); (NCETFJ- 2007); (2008A002) 作者简介 : (1972 ),, ; ; gmliu@jmu.edu.cn 通讯作者 :, mjcao@jmu.edu.cn

kd,,, [7] [8] 48.7 74.4 kd, (Scylla serrata),,,,,,,,,,,,,,, 1 1.

2 1 : 109 表 1 本文实验所用的水产品 Tab. 1 Aquatic products used in this study Mud crab Scylla serrata Chinese mitten crab Eriocheir sinensis Japanese blue crab Portunus trituberculatus White-leg shrimp Penaeus vannamei Grass prawn Penaeus monodon Kuruma prawn Penaeus japonicus Abalone Haliotis diversicolor Short necked clam Ruditapes philippinarum Razor clam Sinonovacula constricta Moerella iridescens Pacific Oyster Crassostrea gigas Argentine shortfin squid Illex argentinus Common carp Cyprinus carpio 1.2 [9] 5 g, 4 10 mmol/l (ph 7.0, 3% NaCl), ( 30s, 1min) min, r/min 20min, 1.3 SDS-PAGE [10] 1.4 Western-blot Dot-blot Western-blot Dot-blot [10] (v/w) 20 mmol/l (ph 7.5)( 50 mmol/l KCl) ( 30s, 1min), 7000 r/min 10min,, 10,,,, mol/l KCl 20 mmol/l (ph 7.5), 1.6 Liang, et al. [11], 1.7 [12] 100 μg 2 kg 300 μl, 300 μl (, ),, ;, ;, 1, 5d ; 37 1h 4,, [13], SDS-PAGE, NC,, HRP IgG, DAB, % NaCl, 12% SDS-PAGE ( 1), 15, kd, kd, 38 kd 50 kd 50% 30% 2.2, min, ( 2),, ( 3 ); kd

monodon Kuruma prawn Penaeus japonicus Abalone Haliotis diversicolor Short necked clam Ruditapes philippinarum Razor clam Sinonovacula constricta Moerella iridescens Pacific Oyster Crassostrea gigas

3 110 34, 38 kd,, IgE 1 Fig. 1 SDS-PAGE analysis of cooked crude extract from Mud crab M. protein marker; 1. cooked crude extract of Mud crab 3 Fig. 3 Western-blot analysis of allergenicity of sarcoplasmic protein from Mud crab M. Prestained Protein Marker; Sera from subjects with crustacean allergy (1 11, respectively); Normal control sera pool 2 Fig. 2 SDS-PAGE analysis of proteins from Mud crab with or without heating M. protein marker; 1. sarcoplasmic protein; 2. myofibrillar protein; 3. supernatant of cooked myogen; 4. supernatant of cooked myofibrillar protein ( 3), 175 kd ( 1 8), 40 kd ( 1 2), ( 9 11)( 12 14) ( 4), 38 kd ( 1 10) 100 kd ( 2 11), kd 4 Fig. 4 Western-blot analysis of allergenicity of myofibrillar protein from Mud crab M. Prestained Protein Marker; Sera from subjects with crustacean allergy (1 11, respectively); 12. Normal control sera pool mol/l KCl, SDS-PAGE 5 2, ( 1), 200 kd 90 kd, 45 kd, 1 mol/l HCl ph, 1 Dot-blot, ph 6.0, ; ph 5.0, ; ph 4.5, ; ph, ph 3, ;

Normal control sera pool 2 Fig. 2 SDS-PAGE analysis of proteins from Mud crab with or without heating M. protein marker; 1. sarcoplasmic protein; 2. myofibrillar protein; 3.

4 1 : 111 ph 4.5 SDS-PAGE 5 3, kd,, , Protein A Sepharose IgG, SDS-PAGE IgG, 50 kd 25 kd 6 Fig. 6 Dot-blot analysis of the purified allergen by Dot-blot Left, serum 1 from subject with crustacean allergy; Right, Normal control sera pool 5 Fig. 5 SDS-PAGE analysis of purification of major allergen from Mud crab M. protein marker; 1. myofibrillar protein; 2. supernatant of salting in; 3. isoelectric point precipitation of ph 4.5; 4. precipitation of 40% 60% ammonium sulfate fractionation; 5. supernatant of cooked precipitation after ammonium sulfate fractionation 20%, 10%, 1 Dot-blot, 30%, ; 40%, ; 50% 60%, ; 70%, ; 40% 60% SDS-PAGE 5 4, 40% 60% 38 kd % 60% min, SDS-PAGE 5 5, 19 kd 100 kd, 38 kd ; Dot-blot ( 6), 1,, 38 kd kd, Western-blot,, ( 7), kd,, ; 7 Fig. 7 SDS-PAGE analysis of crude cooked extracts from different species M. protein marker; 1. Mud crab; 2. Chinese mitten crab; 3. White-leg shrimp; 4. Kuruma prawn; 5. Grass prawn; 6. Abalone; 7. Short necked clam; 8. Razor clam; 9. Moerella iridescens; 10. Pacific Oyster; 11. Argentine shortfin squid; 12. Common carp 38 kd 1 12 Westernblot ( 8),, ;,, ;, 38 kd, 35 kd, 8

$precipitation of 40% 60% ammonium sulfate fractionation; 5.$

5 kd 1 ( 2),, kd,,, kd, 8 38 kd Fig. 8 Western-blot analysis of crude cooked extracts from different species with 38 kd-protein antibody M. protein marker; 1. Mud crab; 2. Chinese mitten crab; 3. White-leg shrimp; 4. Kuruma prawn; 5. Grass prawn; 6. Abalone; 7. Short necked clam; 8. Razor clam; 9. Moerella iridescens; 10. Pacific Oyster; 11. Argentine shortfin squid; 12. Common carp 3,,,,, [8] kd,,,,,, 38 kd, [4-6],,,,,, 40 kd, [14], Tab. 2 表 2 采用甲壳类过敏者血清的免疫印迹检测不同水产品加热粗提物 Western-blot analysis of crude cooked extracts from different species with the sera of crustacean allergic patients Species Number of color band Molecular weight of color band (kd) Mud crab Chinese mitten crab White-leg shrimp Kuruma prawn Grass prawn Abalone 1 36 Short necked clam Razor clam Moerella iridescens Pacific Oyster Argentine shortfin squid Common carp 0 /, pi 4.5, 40% 60%, Pen a 1 [5] [15],, min, SDS- PAGE,, 38 kd -, 38 kd, 4,

Argentine shortfin squid; 12. Common carp 3,,,,, [8] 48.7 74.4 kd,,,,,, 38 kd, [4-6],,,,,, 40 kd, [14], Tab.

6 1 : 113, ; Protein A Sepharose IgG,,, 12, Western-blot,,, kd,,,,,,, 致谢 : : [1] Lehrer S B, Ayuso R, Reese G. Seafood Allergy and Allergens: A Review [J]. Marine Biotechnology (NY), 2003, 5(4): [2] Lü X Z, Liu X M, Yang X G. Preliminary survey on status of food allergy in young Chinese student [J]. Chinese Journal of Food Hygiene, 2005, 17(2): [,,.., 2005, 17(2): ] [3] Food and Agriculture Organization of the United Nations (FAO). Report of the FAO technical consultation on food allergies [M]. Rome, Italy. 1995, [4] Shanti K N, Martin B M, Nagpal S, et al. Identification of tropomyosin as the major shrimp allergen and characterization of its IgE binding epitopes [J]. Journal of Immunology, 1993, 151(10): [5] Daul C B, Slattery M, Reese G, et al. Identification of the major brown shrimp (Penaeus aztecus) allergen as the muscle protein tropomyosin [J]. International Archives of Allergy and Immunology, 1994, 105(1): [6] Leung P S, Chen Y, Gershwin M R, et al. Identification and molecular characterization of Charybdis feriatus tropomyosin, the major crab allergen [J]. Journal of Allergy Clinical Immunology, 1998, 102(5): [7] Motoyama K, Suma Y, Ishizaki S, et al. Molecular cloning of tropomyosins identified as allergens in six species of crustaceans [J]. Journal of Agricultural and Food Chemistry, 2007, 55(3): [8] Zhao Q H, Wang X Z, Chen L J, et al. Purification, identification and characterization of the major allergen from Linnaeus [J]. Chinese Journal of Immunology, 2007, 23(3): [,,,.., 2007, 23(3): ] [9] Li F, Wu H Q, Ge Z C, et al. Comparison of Five Extraction Methods by Analyzing the Total Proteins from Carp Using EMKC [J]. Acta Hydrobiologica Sinica, 2008, 32(1): [,,,.., 2008, 32(1): ] [10] Ausubel F, Short protocols in molecular biology (4th ed) [M]. Beijing: Science Press [. (). :. 2005] [11] Liang Y L, Cao M J, Su W J, et al. Identification and characterization of the major allergen of the Chinese mitten crab (Eriocheir sinensis) [J]. Food Chemistry, 2008, 111(4): [12] Du X L, Liang Y L, Wang X C, et al. Separation, purification and antibody preparation of α-actinin from Skeletal Muscle of Sparus Latus [J]. Journal of Chinese Institute of Food Science and Technology, 2007, 7(4): 8 12 [,,,. α-., 2007, 7(4): 8 12] [13] Joseph S, David W R. Molecular cloning: A laboratory manual (3rd ed) [M]. Beijing: Science Press [. (). :. 2002] [14] Yu C J, Lin Y F, Chiang B L, et al. Proteomics and immunological analyses of a novel shrimp allergen, Pen m 2 [J]. Journal of Immunology, 2003, 170(1): [15] Leung P S, Chu K H, Chow W K, et al. Cloning, expression, and primary structure of Metapenaeus ensis tropomyosin, the major heat-stable shrimp allergen [J]. Journal of Allergy Clinical Immunology, 1994, 94(5):

Chinese Journal of Food Hygiene, 2005, 17(2): 119 121 [,,.., 2005, 17(2): 119 121] [3] Food and Agriculture Organization of the United Nations (FAO).

7 PURIFICATION AND IDENTIFICATION OF THE MAJOR ALLERGEN OF MUD CRAB LIU Guang-Ming, LIANG Yin-Long, WENG Ling, SU Wen-Jin, HUANG Yuan-Yuan and CAO Min-Jie (College of Biological Engineering, Key Laboratory of Science and Technology for Aquaculture and Food Safety in Fujian Province, Jimei University, Xiamen ) Abstract: IgE-mediated hypersensitive reactions to ingestion of crustacean are among the most serious forms of food allergies. Sensitized individuals can develop urticaria, angioedema, asthma, and even life-threatening anaphylaxis. Mud crab (Scylla serrata) is a kind of crab constitutes a promising fishery industry in China. Because of the increasing consumption of such kind of crab, the occurrence of hypersensitive reactions is extending year by year. Although tropomyosin is assumed to be a major allergen in crustaceans, few experimental data are available on allergens in crabs in China, such as Mud crab. Thus, it is important to confirm whether tropomyosin is the major allergen of Mud crab or not. In order to characterize and confirm the biochemical quality of Mud crab allergen, we report herein the isolation, identification, and determination of the major allergen of Mud crab. Crude cooked extract of Mud crab muscle was used as antigen and sera samples from 11 crustacean-allergic patients used as antibody investigated by Western-blot. Allergen with molecular mass about 38 kd was detected by all of 11 sera with crustacean allergy by Western-blot, suggesting this protein was the major allergen of Mud crab. The 38 kd protein was purified to homogeneity by isoelectric point precipitation, ammonium sulfate fractionation, and heating, and it was further characterized as tropomyosin. Polyclonal antibody against Mud crab-tropomyosin was prepared and further purified by Protein A Sepharose affinity column. The antibody cross reacted positively with tropomyosins from other crustaceans, suggesting its potential application in the detecting of the major allergen tropomyosin in crustacean foods. The identification and characterization of the major allergen in Mud crab will facilitate not only the elucidation of cross-reactions to crustaceans but also the advance of the diagnosis and treatment of seafood allergy. The 38 kd tropomyosin allergen in Mud crab as identified in the present study will benefit further allergic studies not only in this species of crab but also in other species of aquatic products. Key words: Mud crab; Allergen; Tropomyosin; Purification

are among the most serious forms of food allergies. Sensitized individuals can develop urticaria, angioedema, asthma, and even life-threatening anaphylaxis.

基础研究食品科学 2011, Vol. 32, No. 07 17 白球蛋白醇溶蛋白和碱溶蛋白等, 发现白果中的蛋白质主要以清蛋白和球蛋白为主, 其中白果清蛋白具有抗氧化和延缓衰老等作

基础研究食品科学 2011, Vol. 32, No. 07 17 白球蛋白醇溶蛋白和碱溶蛋白等, 发现白果中的蛋白质主要以清蛋白和球蛋白为主, 其中白果清蛋白具有抗氧化和延缓衰老等作 16 李莹莹 1, 吴彩娥 1, *, 杨剑婷 2, 范龚健 1 (1. 南京林业大学森林资源与环境学院, 江苏南京 210037;2. 安徽科技学院食品药品学院, 安徽凤阳 233100) 对白果的主要过敏蛋白进行鉴定, 并对其过敏原性进行分析以 SDS-PAGE 和 Western-Blotting

1 : 109 表 1 本 文 实 验 所 用 的 水 产 品 Tab. 1 Aquatic products used in this study Mud crab Scylla serrata Chinese mitten crab Eriocheir sinensis Japanese blu

1 : 109 表 1 本文实验所用的水产品 Tab. 1 Aquatic products used in this study Mud crab Scylla serrata Chinese mitten crab Eriocheir sinensis Japanese blu