牡蛎主要过敏原原肌球蛋白的重组表达及鉴定
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摘要
牡蛎的主要过敏原是Crag 1蛋白(属于一种原肌球蛋白),克隆牡蛎原肌球蛋白Crag 1基因,导入大肠杆菌(BL21)中,然后筛选重组菌株。通过SDS-PAGE探究了重组菌株中Crag 1蛋白的最佳诱导表达条件,15℃下诱导16h。然后通过Western印迹、SDS-PAGE、质谱(MALDI-TOF-MS)、圆二色(CD)光谱等方法从His-tag、相对分子量、肽段比对以及二级结构上综合鉴定了重组Crag 1蛋白的准确性。本实验成功重组表达并鉴定了牡蛎主要过敏原Crag 1蛋白,为接下来的牡蛎脱敏实验以及牡蛎过敏的临床诊断和治疗奠定了良好的生物学基础。
The main allergen of oysters is Crag 1 protein,which is belonged to tropomyosin. Oyster Crag1 gene was cloned into E. coli( BL21),and the recombinant strain was screened. The optimal induction expression conditions of Crag1 protein in recombinant strains were investigated by SDS-PAGE,which was induced at 15℃ for 16 h. Through Western blot,SDS-PAGE,mass spectrometry( MALDI-TOF-MS),circular dichroism( CD)spectroscopy,the accuracy of recombinant Crag 1 protein was comprehensively identified from His-tag,relative molecular weight,peptide alignment and secondary structure. This experiment successfully expressed and identified the oyster main allergen Crag 1 protein,which laid a good biological foundation for future oyster desensitization experiment and clinical diagnosis and treatment of oyster allergy.
The main allergen of oysters is Crag 1 protein,which is belonged to tropomyosin. Oyster Crag1 gene was cloned into E. coli( BL21),and the recombinant strain was screened. The optimal induction expression conditions of Crag1 protein in recombinant strains were investigated by SDS-PAGE,which was induced at 15℃ for 16 h. Through Western blot,SDS-PAGE,mass spectrometry( MALDI-TOF-MS),circular dichroism( CD)spectroscopy,the accuracy of recombinant Crag 1 protein was comprehensively identified from His-tag,relative molecular weight,peptide alignment and secondary structure. This experiment successfully expressed and identified the oyster main allergen Crag 1 protein,which laid a good biological foundation for future oyster desensitization experiment and clinical diagnosis and treatment of oyster allergy.
引文
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[13]Yadzir Z H M,Misnan R,Bakhtiar F,et al.Tropomyosin,the major tropical oyster Crassostrea belcheri allergen and effect of cooking on its allergenicity[J].Allergy,Asthma&Clinical Immunology,11,1(2015-10-26),2015,11(1):30.
[14]Minakata S,Maeda K,N,Wakabayashi K,et al.Twocrystal structures of tropomyosin C-terminal fragment176-273:exposure of the hydrophobic core to the solvent destabilizes the tropomyosin molecule[J].Biophysical Journal,2008,95(2):710.
[2]Dalal I,Binson I,Reifen R,et al.Food allergy is a matter of geography after all:sesame as a major cause of severe IgE-mediated food allergic reactions among infants and young children in Israel[J].Allergy,2002,57(4):362-365.
[3]滕瑜,王彩理.牡蛎的营养和降糖作用研究[J].渔业科学进展,2005,26(6):39-44.
[4]Lopata A L,Kleine-Tebbe J,Kamath S D.Allergens and molecular diagnostics of shellfish allergy:Part 22 of the Series Molecular Allergology[J].Allergo Journal International,2016,25(7):210-218.
[5]Thammathongchat S,Hagiwara T,Sakiyama T.Adsorption of tropomyosin from pink shrimp(Pandalus eous)on stainless steel surface[J].Food Control,2010,21(9):1250-1253.
[6]李启沅,李荔,刘志刚.鱼虾贝类的主要过敏原分子免疫学特性研究进展[J].水产科学,2008,27(3):154-156.
[7]Leung P S,Chu K H.cDNA cloning and molecular identification of the major oyster allergen from the Pacific oyster Crassostrea gigas[J].Clinical&Experimental Allergy,2010,31(8):1287-1294.
[8]Ishikawa M,Ishida M,Shimakura K,et al.Tropomyosin,the major oyster Crassostrea gigas allergen and its IgE-binding epitopes[J].Journal of Food Science,2010,63(1):44-47.
[9]Lopata A L,Kamath S.Shellfish allergy diagnosis-gaps and needs[J].Current Allergy&Clinical Immunology,2012,25(2):60-66.
[10]张雅丽,杨国庆,郭英,等.密码子优化的牛精蛋白基因在大肠杆菌中的表达[J].高技术通讯,2002,12(4):42-46.
[11]Reese G,Ayuso R,Lehrer S B.Tropomyosin:an invertebrate pan-allergen[J].International Archives of Allergy&Immunology,1999,119(4):247-258.
[12]黄榕芳.软体类海产品主要过敏原的基因克隆及生物信息学分析[D].中国海洋大学,2014.
[13]Yadzir Z H M,Misnan R,Bakhtiar F,et al.Tropomyosin,the major tropical oyster Crassostrea belcheri allergen and effect of cooking on its allergenicity[J].Allergy,Asthma&Clinical Immunology,11,1(2015-10-26),2015,11(1):30.
[14]Minakata S,Maeda K,N,Wakabayashi K,et al.Twocrystal structures of tropomyosin C-terminal fragment176-273:exposure of the hydrophobic core to the solvent destabilizes the tropomyosin molecule[J].Biophysical Journal,2008,95(2):710.