花生过敏原Ara h 1蛋白的原核表达及致敏性分析
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摘要
从花生中提取总RNA,用反转录聚合酶链式反应得到花生过敏原Ara h 1基因,构建pET-28a-Ara h 1表达载体,转入Rosetta(DE3)宿主表达菌中诱导产物表达,用镍离子亲和层析法纯化得到目的蛋白。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳结果显示目的蛋白分子质量约为75 kDa,与预计相符;经质谱鉴定为Ara h 1蛋白。用BALB/c小鼠模型评价重组Ara h 1蛋白的致敏性结果显示,重组Ara h 1蛋白致敏小鼠血清中特异性抗体、Th2型细胞因子、组胺含量升高,空肠和肺组织发生病变,表明重组Ara h 1蛋白可以导致小鼠发生Th2型过敏反应,且有与天然Ara h 1蛋白相似的致敏性。同时RBL细胞模型结果显示重组Ara h 1蛋白还可导致RBL细胞脱颗粒,释放β-己糖苷酶,进一步表明重组Ara h 1蛋白具有致敏性。
An Ara h 1 gene was synthesized from total RNA extracted from peanut using oligo primers by reverse transcription polymerase chain reaction(RT-PCR). The gene was cloned into pET-28 a vector and a recombinant expression vector was constructed. Then the recombinant vector was transformed into the bacterial strain Rosetta(DE3) for protein expression, and the target protein was puri?ed by Ni af?nity chromatography. Sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE) showed that the molecular mass of the expressed protein was about 75 kDa, which matched with the theoretical value. Analysis by mass spectrometry(MS) con?rmed that the protein was Ara h 1. The allergenicity of recombinant Ara h 1 protein was evaluated using an animal model of BALB/c mice. The results showed that recombinant Ara h 1 protein had a similar allergenicity to the native one. The contents of specific antibody, Th2 type cytokines and histamine in the serum were increased, and in?ammation occurred in the lung and jejunum of mice sensitized to recombinant Ara h 1. Meanwhile, recombinant Ara h 1 could trigger degranulation of RBL cell, resulting in release of β-hexosaminidase.Accordingly, recombinant Ara h 1 could cause Th2 type allergic reaction.
An Ara h 1 gene was synthesized from total RNA extracted from peanut using oligo primers by reverse transcription polymerase chain reaction(RT-PCR). The gene was cloned into pET-28 a vector and a recombinant expression vector was constructed. Then the recombinant vector was transformed into the bacterial strain Rosetta(DE3) for protein expression, and the target protein was puri?ed by Ni af?nity chromatography. Sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE) showed that the molecular mass of the expressed protein was about 75 kDa, which matched with the theoretical value. Analysis by mass spectrometry(MS) con?rmed that the protein was Ara h 1. The allergenicity of recombinant Ara h 1 protein was evaluated using an animal model of BALB/c mice. The results showed that recombinant Ara h 1 protein had a similar allergenicity to the native one. The contents of specific antibody, Th2 type cytokines and histamine in the serum were increased, and in?ammation occurred in the lung and jejunum of mice sensitized to recombinant Ara h 1. Meanwhile, recombinant Ara h 1 could trigger degranulation of RBL cell, resulting in release of β-hexosaminidase.Accordingly, recombinant Ara h 1 could cause Th2 type allergic reaction.
引文
[1]SAMPSON H A.Food allergy:when mucosal immunity goes wrong[J].Journal of Allergy and Clinical Immunology,2005,115(1):139-141.DOI:10.1016/j.jaci.2004.11.003.
[2]陈红兵,高金燕.食物过敏反应及其机制[J].营养学报,2007(2):105-109.
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[8]OSTERBALLE M,HANSEN T K,MORTZ C G,et al.The prevalence of food hypersensitivity in an unselected population of children and adults[J].Pediatric Allergy and Immunology,2005,16(7):567-573.
[9]常雪娇,李坤,张英,等.晒干处理对花生过敏原蛋白潜在致敏性的影响[J].食品科学,2018,39(3):49-54.DOI:10.7506/spkx1002-6630-201803008.
[10]PARK C W,KIM G I,LEE C H.A comparison study on allergen components between Korean(Arachis fastigiata Shinpung)and American peanut(Arachis hypogaea Runner)[J].Journal of Korean Medical Science,2000,15(4):387-392.DOI:10.3346/jkms.2000.15.4.387.
[11]罗春萍,高金燕,胡纯秋,等.花生交叉过敏反应的研究进展[J].食品科学,2009,30(21):384-388.
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[13]LORENZ A R,SCHEURER S,HAUSTEIN D,et al.Recombinant food allergens[J].Journal of Chromatography B,2001,756(1/2):255-279.DOI:10.1016/S0378-4347(01)00086-X.
[14]VALENTA R,NIEDERBERGER V.Recombinant allergens for immunotherapy[J].Journal of Allergy and Clinical Immunology,2007,119(4):826-830.DOI:10.1016/j.jaci.2007.01.025.
[15]高琳,杨安树,高金燕,等.食物过敏原致敏性评估方法研究进展[J].食品科学,2014,35(7):252-257.DOI:10.7506/spkx1002-6630-201407050.
[16]黄建芳,王彩霞,向军俭,等.食物过敏动物模型的研究进展[J].食品科学,2014,35(3):280-284.DOI:10.7506/spkx1002-6630-201403055.
[17]MOGHADDAM A E,HILLSON W R,NOTI M,et al.Dry roasting enhances peanut-induced allergic sensitization across mucosal and cutaneous routes in mice[J].Journal of Allergy and Clinical Immunology,2014,134(6):1453-1456.DOI:10.1016/j.jaci.2014.07.032.
[18]PASSANTE E,FRANKISH N.The RBL-2H3 cell line:its provenance and suitability as a model for the mast cell[J].Inflammation Research,2009,58(11):737-745.DOI:10.1007/s00011-009-0074-y.
[19]罗霞,王青,周联,等.几种常见中药注射剂对RBL-2H3细胞脱颗粒的影响[J].中药新药与临床药理,2009,20(6):506-510.
[20]SUN N,ZHOU C,ZHOU X,et al.Use of a rat basophil leukemia(RBL)cell-based immunological assay for allergen identification,clinical diagnosis of allergy,and identification of anti-allergy agents for use in immunotherapy[J].Journal of Immunotoxicology,2015,12(2):199-205.DOI:10.3109/1547691X.2014.920063.
[21]SHI X,GUO R,WHITE B L,et al.Allergenic properties of enzymatically hydrolyzed peanut flour extracts[J].International Archives of Allergy and Immunology,2013,162(2):123-130.DOI:10.1159/000351920.
[22]CHRUSZCZ M,MALEKI S J,MAJOREK K A,et al.Structural and immunologic characterization of Ara h 1,a major peanut allergen[J].Journal of Biological Chemistry,2011,286(51):44294-44294.DOI:10.1074/jbc.A111.270132.
[23]RAMOS M L,HUNTLEY J J,MALEKI S J,et al.Identification and characterization of a hypoallergenic ortholog of Ara h 2.01[J].Plant Molecular Biology,2009,69(3):325-335.DOI:10.1007/s11103-008-9428-z.
[24]詹少德,邱昌将,朱盼,等.花生过敏原蛋白Ara h 6基因克隆和原核表达[J].食品科学,2016,37(3):125-130.DOI:10.7506/spkx1002-6630-201603024.
[25]刘志刚,杨成彬,闫浩,等.Balb/c小鼠花生过敏模型的建立及发病机理[J].深圳大学学报(理工版):2012,29(2):165-170.
[26]ZHANG W J,ZHU Q Q,ZHANG T,et al.Thermal processing effects on peanut allergen Ara h 2 allergenicity in mice and its antigenic epitope structure[J].Food Chemistry,2016,212(1):657-662.DOI:10.1016/j.foodchem.2016.06.036.
[27]陈同强,郭丹,邱立明,等.龙虾过敏小鼠模型建立的研究[J].赣南医学院学报,2013,33(6):815-818.
[28]MOSMANN T R,COFFMAN R L.TH1 and TH2 cells:different patterns of lymphokine secretion lead to different functional properties[J].Annual Review of Immunology,1989,7(1):145-173.DOI:10.1146/annurev.iy.07.040189.001045.
[29]WANG J,SAMPSON H A.Food allergy:recent advances in pathophysiology and treatment[J].Allergy Asthma&Immunology Research,2009,1(1):19-29.DOI:10.4168/aair.2009.1.1.19.
[30]YAMASAKI S,SAITO T.Progress in allergy signal research on mast cells:signal regulation of multiple mast cell responses through Fc epsilon RI[J].Journal of Pharmacological Sciences,2008,106(3):336-340.DOI:10.1254/jphs.FM0070251.
[31]孙娜,周催,王翠燕,等.大鼠嗜碱性白血病粒细胞系RBL-2H3和RBL-1细胞模型在评价食品潜在致敏性中的比较[J].农业生物技术学报,2013,21(12):1465-1472.
[2]陈红兵,高金燕.食物过敏反应及其机制[J].营养学报,2007(2):105-109.
[3]PANTEGHINI M,BUNK D M,CHRISTENSON R H,et al.Standardization of troponin I measurements:an update[J].Clinical Chemistry and Laboratory Medicine,2008,46(11):1501-1506.DOI:10.1515/Cclm.2008.291.
[4]FERNANDEZ-RIVAS M,BOLHAAR S,GONZALEZ-MANCEBO E,et al.Apple allergy across Europe:how allergen sensitization profiles determine the clinical expression of allergies to plant foods[J].Journal of Allergy and Clinical Immunology,2006,118(2):481-488.DOI:10.1016/j.jaci.2006.05.012.
[5]ZHANG W J,CAI Q,GUAN X,et al.Detection of peanut(Arachis hypogaea)allergen by real-time PCR method with internal amplification control[J].Food Chemistry,2015,174(1):547-552.DOI:10.1016/j.foodchem.2014.11.091.
[6]SICHERER S H,MUNOZ-FURLONG A,SAMPSON H A.Prevalence of peanut and tree nut allergy in the United States determined by means of a random digit dial telephone survey:a 5-year follow-up study[J].Allergy Clinical Immunology,2003,112(6):1203-1207.
[7]MORISSET M,MONERET-VAUTRIN D A,KANNY G.Prevalence of peanut sensitization in a population of 4,737 subjects:an Allergo Vigilance Network enquiry carried out in 2002[J].Allergy Immunology,2005,37(2):54-57.
[8]OSTERBALLE M,HANSEN T K,MORTZ C G,et al.The prevalence of food hypersensitivity in an unselected population of children and adults[J].Pediatric Allergy and Immunology,2005,16(7):567-573.
[9]常雪娇,李坤,张英,等.晒干处理对花生过敏原蛋白潜在致敏性的影响[J].食品科学,2018,39(3):49-54.DOI:10.7506/spkx1002-6630-201803008.
[10]PARK C W,KIM G I,LEE C H.A comparison study on allergen components between Korean(Arachis fastigiata Shinpung)and American peanut(Arachis hypogaea Runner)[J].Journal of Korean Medical Science,2000,15(4):387-392.DOI:10.3346/jkms.2000.15.4.387.
[11]罗春萍,高金燕,胡纯秋,等.花生交叉过敏反应的研究进展[J].食品科学,2009,30(21):384-388.
[12]SHIN D,SAMPSON H A,HUANG S K,et al.Characterization of a major peanut allergen:mutational analysis of the Ara h 1 IgE binding epitopes[J].Journal of Allergy and Clinical Immunology,1997,99(1):570-570.
[13]LORENZ A R,SCHEURER S,HAUSTEIN D,et al.Recombinant food allergens[J].Journal of Chromatography B,2001,756(1/2):255-279.DOI:10.1016/S0378-4347(01)00086-X.
[14]VALENTA R,NIEDERBERGER V.Recombinant allergens for immunotherapy[J].Journal of Allergy and Clinical Immunology,2007,119(4):826-830.DOI:10.1016/j.jaci.2007.01.025.
[15]高琳,杨安树,高金燕,等.食物过敏原致敏性评估方法研究进展[J].食品科学,2014,35(7):252-257.DOI:10.7506/spkx1002-6630-201407050.
[16]黄建芳,王彩霞,向军俭,等.食物过敏动物模型的研究进展[J].食品科学,2014,35(3):280-284.DOI:10.7506/spkx1002-6630-201403055.
[17]MOGHADDAM A E,HILLSON W R,NOTI M,et al.Dry roasting enhances peanut-induced allergic sensitization across mucosal and cutaneous routes in mice[J].Journal of Allergy and Clinical Immunology,2014,134(6):1453-1456.DOI:10.1016/j.jaci.2014.07.032.
[18]PASSANTE E,FRANKISH N.The RBL-2H3 cell line:its provenance and suitability as a model for the mast cell[J].Inflammation Research,2009,58(11):737-745.DOI:10.1007/s00011-009-0074-y.
[19]罗霞,王青,周联,等.几种常见中药注射剂对RBL-2H3细胞脱颗粒的影响[J].中药新药与临床药理,2009,20(6):506-510.
[20]SUN N,ZHOU C,ZHOU X,et al.Use of a rat basophil leukemia(RBL)cell-based immunological assay for allergen identification,clinical diagnosis of allergy,and identification of anti-allergy agents for use in immunotherapy[J].Journal of Immunotoxicology,2015,12(2):199-205.DOI:10.3109/1547691X.2014.920063.
[21]SHI X,GUO R,WHITE B L,et al.Allergenic properties of enzymatically hydrolyzed peanut flour extracts[J].International Archives of Allergy and Immunology,2013,162(2):123-130.DOI:10.1159/000351920.
[22]CHRUSZCZ M,MALEKI S J,MAJOREK K A,et al.Structural and immunologic characterization of Ara h 1,a major peanut allergen[J].Journal of Biological Chemistry,2011,286(51):44294-44294.DOI:10.1074/jbc.A111.270132.
[23]RAMOS M L,HUNTLEY J J,MALEKI S J,et al.Identification and characterization of a hypoallergenic ortholog of Ara h 2.01[J].Plant Molecular Biology,2009,69(3):325-335.DOI:10.1007/s11103-008-9428-z.
[24]詹少德,邱昌将,朱盼,等.花生过敏原蛋白Ara h 6基因克隆和原核表达[J].食品科学,2016,37(3):125-130.DOI:10.7506/spkx1002-6630-201603024.
[25]刘志刚,杨成彬,闫浩,等.Balb/c小鼠花生过敏模型的建立及发病机理[J].深圳大学学报(理工版):2012,29(2):165-170.
[26]ZHANG W J,ZHU Q Q,ZHANG T,et al.Thermal processing effects on peanut allergen Ara h 2 allergenicity in mice and its antigenic epitope structure[J].Food Chemistry,2016,212(1):657-662.DOI:10.1016/j.foodchem.2016.06.036.
[27]陈同强,郭丹,邱立明,等.龙虾过敏小鼠模型建立的研究[J].赣南医学院学报,2013,33(6):815-818.
[28]MOSMANN T R,COFFMAN R L.TH1 and TH2 cells:different patterns of lymphokine secretion lead to different functional properties[J].Annual Review of Immunology,1989,7(1):145-173.DOI:10.1146/annurev.iy.07.040189.001045.
[29]WANG J,SAMPSON H A.Food allergy:recent advances in pathophysiology and treatment[J].Allergy Asthma&Immunology Research,2009,1(1):19-29.DOI:10.4168/aair.2009.1.1.19.
[30]YAMASAKI S,SAITO T.Progress in allergy signal research on mast cells:signal regulation of multiple mast cell responses through Fc epsilon RI[J].Journal of Pharmacological Sciences,2008,106(3):336-340.DOI:10.1254/jphs.FM0070251.
[31]孙娜,周催,王翠燕,等.大鼠嗜碱性白血病粒细胞系RBL-2H3和RBL-1细胞模型在评价食品潜在致敏性中的比较[J].农业生物技术学报,2013,21(12):1465-1472.