牙鲆(Paralichthys olivaceus)中不同亚型小清蛋白的提纯与鉴定
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摘要
以牙鲆(Paralichthys olivaceus)为研究对象,以Tris、甘氨酸和二硫苏糖醇的混合溶液为提取液,加入不同量的硫酸铵分级纯化小清蛋白,应用免疫印迹法、酶联免疫法及质谱法进行鉴定。最终提纯得到3种不同分子质量的蛋白(PVⅠ、PVⅡ和PVⅢ),所得蛋白纯度大于90%,血清学实验证明其与小清蛋白特异性IgG及IgE抗体均有结合能力,质谱鉴定得到PVII和PVIII分子质量分别为12 151 Da和11 645 Da,pI值分别为5.14和4.69。该方法满足牙鲆小清蛋白提取纯化要求,得到的3种蛋白是牙鲆小清蛋白的不同亚型,且均具有过敏原性。
Parvalbumins were extracted and purified from the muscle of Japanese flounder(Paralichthys olivaceus). A mixed solution of Tris, glycine, and DL-dithiothreitol was used as the extraction solvent. Parvalbumins were purified by adding ammonium sulfate to different degrees of saturation, and then identified using enzyme-linked immunosorbent assay(ELISA), Western-blot and high performance liquid chromatography-tandem mass spectrometry(HPLC-MS/MS). Three proteins(PVI, PVII and PVIII) with different molecular masses were obtained with high purity( > 90%). Serological results demonstrated that each of these parvalbumins had the ability of binding to the specific IgG and IgE. The molecular masses of PVII and PVIII were determined to be 12 151 and 11 645 Da with Isoelectric point(pI) of 5.14 and 4.69, respectively. This method can meet the requirements for the extraction and purification of parvalbumins. These proteins were proved to represent different isotypes of parvalbumins with allergenicity.
Parvalbumins were extracted and purified from the muscle of Japanese flounder(Paralichthys olivaceus). A mixed solution of Tris, glycine, and DL-dithiothreitol was used as the extraction solvent. Parvalbumins were purified by adding ammonium sulfate to different degrees of saturation, and then identified using enzyme-linked immunosorbent assay(ELISA), Western-blot and high performance liquid chromatography-tandem mass spectrometry(HPLC-MS/MS). Three proteins(PVI, PVII and PVIII) with different molecular masses were obtained with high purity( > 90%). Serological results demonstrated that each of these parvalbumins had the ability of binding to the specific IgG and IgE. The molecular masses of PVII and PVIII were determined to be 12 151 and 11 645 Da with Isoelectric point(pI) of 5.14 and 4.69, respectively. This method can meet the requirements for the extraction and purification of parvalbumins. These proteins were proved to represent different isotypes of parvalbumins with allergenicity.
引文
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[23]马涛,王一侠,金振涛,等.三文鱼过敏原小清蛋白分离纯化及免疫结构鉴定[J].食品研究与开发,2017,38(4):1-5.DOI:10.3969/j.issn.1005-6521.2017.04.001.
[24]傅玲琳,富舒洁,王彦波,等.凡纳对虾原肌球蛋白硫酸铵沉淀分离纯化方法的优化[J].食品科学,2017,38(18):187-192.DOI:10.7506/spkx1002-6630-201718030.
[25]龚小玲,鲍宝龙,杨桂梅,等.牙鲆变态早期cDNA文库的构建和parvalbumin基因的克隆[J].水产学报,2005,29(4):467-472.DOI:10.3321/j.issn:1000-0615.2005.04.006.
[26]YUKIHIRO K,TAO Y,CHENG T Y,et al.Quantification of major allergen parvalbumin in 22 species of fish by SDS-PAGE[J].Food Chemistry,2016,194:345-353.DOI:10.1016/j.foodchem.2015.08.037.
[27]KOBAYASHI A,KOBAYASHI Y,SHIOMI K.Fish allergy in patients with parvalbumin-specific immunoglobulin E depends on parvalbumin content rather than molecular differences in the protein among fish species[J].Journal of the Agricultural Chemical Society of Japan,2016,80(10):2018-2021.DOI:10.1080/09168451.2016.1189318.
[28]KUEHN A,SCHEUERMANN T,HILGER C,et al.Important variations in parvalbumin content in common fish species:a factor possibly contributing to variable allergenicity[J].International Archives of Allergy and Immunology,2010,153(4):359-366.DOI:10.1159/000316346.
[29]GRIESMEIER U,VáZQUEZ-CORTéS S,BUBLIN M,et al.Expression levels of parvalbumins determine allergenicity of fish species[J].Allergy,2010,65(2):191-198.DOI:10.1111/j.1398-9995.2009.02162.x.
[30]GUO F F,KUBOTA H,SHIOMI K.Purification,immunological properties and molecular cloning of two allergenic parvalbumins from the crimson sea bream,Evynnis japonica[J].Food Chemistry,2012,132(2):835-840.DOI:10.1016/j.foodchem.2011.11.046.
[2]SHARP M F,LOPATA A L.Fish allergy:in review[J].Clinical Reviews in Allergy&Immunology,2014,46(3):258-271.DOI:10.1007/s12016-013-8363-1.
[3]Commission directive 2000/13/EC of the European Parliament and of the Council of 20 March 2000 on the approximation of the laws of the Member States relating to the labelling,presentation and advertising of foodstuffs,OJ L109[R].2000:29-42.
[4]Commission Directive 2007/68/EC of 27 November 2007 amending Annex IIIa to Directive 2000/13/EC of the European Parliament and of the Council as regards certain food ingredients,OJ L 310[R].2007:11-14.
[5]Regulation(EU)No 1169/2011 of the European Parliament and of the Council of 25 October 2011 on the provision of food information to consumers,OJ L 304,22.11[R].2011:18-63.
[6]FDA.Food allergen labeling and consumer protection act[R].Silver Spring:US Food and Drug Administration,2004.
[7]PEREZGORDO M,CUESTAHERRANZ J,MAROTO A S,et al.Identification of sole parvalbumin as a major allergen:study of cross-reactivity between parvalbumins in a Spanish fish-allergic population[J].Clinical&Experimental Allergy,2011,41(5):750-758.DOI:10.1111/j.1365-2222.2011.03721.x.
[8]CARRERA M,CANAS B,GALLARDO J M.Rapid direct detection of the major fish allergen,parvalbumin,by selected MS/MS ion monitoring mass spectrometry[J].Journal of Proteomics,2012,75(11):3211-3220.DOI:10.1016/j.jprot.2012.03.030.
[9]KUEHN A,SWOBODA I,ARUMUGAM K,et al.Fish allergens at a glance:variable allergenicity of parvalbumins,the major fish allergens[J].Frontiers in Immunology,2014,5:179:1-8.DOI:10.3389/fimmu.2014.00179.
[10]ELSAYED S,AAS K.Isolation of purified allergens(Cod)by isoelectric focusing[J].International Archives of Allergy&Applied Immunology,1971,40(3):428-438.DOI:10.1159/000230425.
[11]BALLMER-WEBER B K,FERNANDEZ-RIVAS M,BEYER K,et al.How much is too much?Threshold dose distributions for 5 food allergens[J].Journal of Allergy and Clinical Immunology,2015,135(4):964-971.DOI:10.1016/j.jaci.2014.10.047.
[12]CAI Q F,LIU G M,LI T,et al.Purification and characterization of parvalbumins,the major allergens in red stingray(Dasyatis akajei)[J].Journal of Agricultural&Food Chemistry,2010,58(24):12964-12969.DOI:10.1021/jf103316h.
[13]HAMADA Y,SATO T A,ISHIZAKI S,et al.Expression and evaluation of IgE-binding capacity of recombinant Pacific mackerel parvalbumin[J].Allergology International,2004,53(3):271-278.DOI:10.1111/j.1440-1592.2004.00344.x.
[14]KOBAYASHI A,KOBAYASHI Y,SHIOMI K.Fish allergy in patients with parvalbumin-specific immunoglobulin E depends on parvalbumin content rather than molecular differences in the protein among fish species[J].Journal of the Agricultural Chemical Society of Japan,2016,80(10):2018-2021.DOI:10.1080/09168451.2016.1189318.
[15]LAEMMLI U K.Cleavage of structural proteins during the assembly of the head of bacteriophage T4[J].Nature,1970,227:680-685.DOI:10.1038/227680a0.
[16]OFFENGENDEN M,FENTABIL M A,WU J.N-glycosylation of ovomucin from hen egg white[J].Glycoconjugate Journal,2011,28(3/4):113-123.DOI:10.1007/s10719-011-9328-3.
[17]SHIOMI K,HAMADA Y,SEKIGUCHI K,et al.Two classes of allergens,parvalbumins and higher molecular weight substances,in Japanese eel[Anguilla japonica]and bigeye tuna[Thunnus obesus][J].Fisheries Science,1999,65(6):943-948.DOI:10.2331/fishsci.65.943.
[18]LI Z,YOU J,LUO Y K,et al.Purification and characterization of parvalbumin isotypes from grass carp(Ctenopharyngodon idella)[J].Journal of Agricultural&Food Chemistry,2014,62(26):6212-6218.DOI:10.1080/09168451.2016.1189318.
[19]BEALE J E,JEEBHAY M F,LOPATA A L.Characterisation of purified parvalbumin from five fish species and nucleotide sequencing of this major allergen from Pacific pilchard,Sardinops sagax[J].Molecular Immunology,2009,46(15):2985-2993.DOI:10.1016/j.molimm.2009.06.018.
[20]LIU R,KRISHNAN H B,XUE W,et al.Characterization of allergens isolated from the freshwater fish blunt snout bream(Megalobrama amblycephala)[J].Journal of Agricultural&Food Chemistry,2011,59(1):458-463.DOI:10.1021/jf103942p.
[21]MA J J,RAMESH P T,LI Z X,et al.Optimisation of an extraction technique of fish allergens suitable for detection and diagnosis[J].Czech Journal of Food Sciences,2017,35(1):24-31.DOI:10.17221/578/2015-CJFS.
[22]ARIF S H,JABEEN M,HASNAIN A U.BIochemical characterization and thermostable capacity of parvalbumins:the major fish-food allergens[J].Journal of Food Biochemistry,2010,31(1):121-137.DOI:10.1111/j.1745-4514.2007.00104.x.
[23]马涛,王一侠,金振涛,等.三文鱼过敏原小清蛋白分离纯化及免疫结构鉴定[J].食品研究与开发,2017,38(4):1-5.DOI:10.3969/j.issn.1005-6521.2017.04.001.
[24]傅玲琳,富舒洁,王彦波,等.凡纳对虾原肌球蛋白硫酸铵沉淀分离纯化方法的优化[J].食品科学,2017,38(18):187-192.DOI:10.7506/spkx1002-6630-201718030.
[25]龚小玲,鲍宝龙,杨桂梅,等.牙鲆变态早期cDNA文库的构建和parvalbumin基因的克隆[J].水产学报,2005,29(4):467-472.DOI:10.3321/j.issn:1000-0615.2005.04.006.
[26]YUKIHIRO K,TAO Y,CHENG T Y,et al.Quantification of major allergen parvalbumin in 22 species of fish by SDS-PAGE[J].Food Chemistry,2016,194:345-353.DOI:10.1016/j.foodchem.2015.08.037.
[27]KOBAYASHI A,KOBAYASHI Y,SHIOMI K.Fish allergy in patients with parvalbumin-specific immunoglobulin E depends on parvalbumin content rather than molecular differences in the protein among fish species[J].Journal of the Agricultural Chemical Society of Japan,2016,80(10):2018-2021.DOI:10.1080/09168451.2016.1189318.
[28]KUEHN A,SCHEUERMANN T,HILGER C,et al.Important variations in parvalbumin content in common fish species:a factor possibly contributing to variable allergenicity[J].International Archives of Allergy and Immunology,2010,153(4):359-366.DOI:10.1159/000316346.
[29]GRIESMEIER U,VáZQUEZ-CORTéS S,BUBLIN M,et al.Expression levels of parvalbumins determine allergenicity of fish species[J].Allergy,2010,65(2):191-198.DOI:10.1111/j.1398-9995.2009.02162.x.
[30]GUO F F,KUBOTA H,SHIOMI K.Purification,immunological properties and molecular cloning of two allergenic parvalbumins from the crimson sea bream,Evynnis japonica[J].Food Chemistry,2012,132(2):835-840.DOI:10.1016/j.foodchem.2011.11.046.