LIN Su-xia,WANG Xiao-mei,LIU Zhi-gang,et al.Cloning and Expression of the Antigenic Epitope of Gly m Bd 30K Protein from Soybean and Purification and Identification of Expressed Product[J].Soybean Science,2010,29(02):186-190.[doi:10.11861/j.issn.1000-9841.2010.02.0186]
大豆主要过敏原GlymBd30K的抗原表位区基因的克隆表达、纯化及免疫原性鉴定
- Title:
- Cloning and Expression of the Antigenic Epitope of Gly m Bd 30K Protein from Soybean and Purification and Identification of Expressed Product
- 文章编号:
- 1000-9841（2010）02-0186-05
- Keywords:
- The major allergens of soybean; Gly m Bd 30k; Epitope; Cloning
- 分类号:
- S565.1
- 文献标志码:
- A
- 摘要:
- 根据文献并结合生物信息学方法选取大豆主要过敏原GlymBd30k的抗原表位区,采用PCR方法扩增出该抗原表位区基因片段,并通过酶切连接分别构建单体的pET表达载体(pET-sGly)和二聚体的pET表达载体(pET-dGly),重组质粒转化到大肠杆菌BL21(DE3)plysS,经IPTG诱导后进行SDS-PAGE分析；同时用Ni2+离子亲和层析柱纯化表达的sGly和dGly抗原表位蛋白,用western-blotting和ELISA检测重组蛋白的免疫原性。结果表明：表达的单体sGly蛋白以可溶性表达为主，而其二聚体dGly蛋白以包涵体形式存在,纯化的sGly和dGly蛋白都具有较好的免疫原性，重组蛋白sGly的抗原性更佳。成功构建的大豆主要过敏原GlymBd30k蛋白的抗原表位区单体sGly蛋白及其二聚体dGly蛋白的工程菌,为研制大豆主要过敏原的单克隆抗体以制备用于大豆主要过敏原检测的试剂奠定了基础。
- Abstract:
- Through reviewing the literatures combined with bioinformatics predication, the antigenic epitope of Gly m Bd 30k allergen was selected. And the gene was amplified by PCR. The pET expression vector for the monomer protein (pET-sGly) and the dimer (pET-dGly) were constructed. The recombinant proteins were expressed in E.coli BL21 (DE3) plysS by IPTG, analyzed by SDS-PAGE, and then purified by metal (Ni2+) chelating affinity chromatography.The immunogenicity was tested by western-blotting and ELISA. Results shows that the monomer protein sGly was mainly expressed as soluble protein, while the dimer protein dGly as inclusion bodies. Purified protein sGly and dGly both have good immunogenicity, and sGly is better. The prokaryotic expression vectors for the monomer protein and dimer of the antigenic epitope of Gly m Bd 30k protein of soybean will make great foundations for developing the monoclonal antibodies and the detection kit of the major allergens of soybean.
参考文献/References:
[1]郑云兰,李霞辉.大豆营养分析技术[M].哈尔滨: 黑龙江科学技术出版社, 1991: 190. (Zheng Y L, Li X H. Soybean nutritional analysis[M]. Harbin: Heilongjiang Science and Technology Publishing House, 1991: 190. ) [2]Duke W W. Soybean as a possible important source of allergy [J].Journal of Allergy,1934, 5:300-302. [3]李钢,丁淑霞,左容,等.食物过敏引起变态反应疾病184例临床分析[J].佳木斯医学院学报,1989, 12 (3) : 269. (Li G, Ding S X, Zuo R, et al. Food allergies cause allergic disease clinical analysis of 184 cases [J]. Jiamusi Medical College, 1989, 12(3): 269. ) [4]徐上浩.进食豆浆引起过敏性休克1例报告[J].海军医学,1992,10(2):185.(Xu S H. A report of Anaphylactic shock caused by the consumption of soy milk[J]. Naval Medical, 1992, 10(2):185.) [5]Ogawa T, Bando N, Tsuji H, et al. Investigation of the IgE binding proteins in soybeans by immunoblotting with the sera of the soybean sensitive patients with atopic dermatitis [J]. Journal of Nutritional Science and Vitaminology, 1991, 37(6): 555-565. [6]Bando N, Tsuji H, Yamanishi R, et al. Identification of the glycosylation site of a major soybean allergen, Gly m Bd 30K[J]. Bioscience, Biotechnology and Biochemistry，1996, 60(2):347-348. 〖JP〗 [7]Hosoyama H, Obata A, Bando N,et al. Epitope analysis of soybean major allergen Gly m Bd 30K recognized by the mouse monoclonal antibody using overlapping peptides[J]. Biosci Biotechnol Biochem, 1996, 60(7):1181-1182. [8]Babiker E E, Azakami H, Ogawa T, et al. Immunological characterization of recombinant soy protein allergen produced by Escherichia coli expression system[J]. Agricultural and Food Chemistry，2000, 48(2):571-575. [9]邬玉兰,刘志刚.大豆主要过敏原Gly m Bd 30K基因的克隆及其原核表达载体的构建[J].大豆科学， 2009, 28 (1):11-15. (Wu Y L, Liu Z G. Cloning and prokaryotic expression vector construction of Gly m Bd 30K gene from soybean(Glycine max)[J]. Soybean Science, 2009, 28(1):11-15.) [10]王莉,刘道杰,李连之.亲和层析在蛋白质研究中的应用进展[J]. 理化检验(化学分册),2007,43(6)：515-517.(Wang L, Liu D J, LI L Z. Advances of application of affinity chromatography to research on protein[J]. Physical Testing and Chemical Analysis(Part B:Chemical Analysis), 2007, 43(6):515-517.) [11]Dean D M, Hugh A S, Ronald A S. Food Allergy: Adverse reactions to foods and food additives[M]. (Second Edition) USA Blackwell Scienceinc，1997:53-67. [12]周淑红.国外关于食品过敏标签的现状及启示[J].世界农业,2007(6):67-68.(Zhou S H. The apocalypse and status on food allergy labels in abroad[J]. World Agriculture, 2007(6):67-68.) [13]陈家杰,朱海,叶卫翔,等.双抗体夹心ELISA法测定食物中大豆过敏原蛋白成分[J].食品研究与开发, 2009,30(5):105-109.(Chen J J, Zhu H, Ye W X, et al. Detection of soybean allergen protein trace in food products by sandwich-antibody enzyme linked immunosorbent assay[J]. Food Research and Developent,2009,30(5):105-109.) [14]游金明,王自蕊,谯仕彦,等.致仔猪过敏性大豆抗原蛋白β-conglycinin单克隆抗体的制备与鉴定[J]. 中国畜牧杂志,2008,44(17):46-49.(You J M, Wang Z R, Qiao S Y, et al. Preparation and characterization of monoclonal antibodies against soybean β-conglycinin, a hypersensitive allergen to piglets[J]. Chinese Journal of Animal Science,2008, 44(17):46-49.)
备注/Memo
基金项目：广东省科技计划资助项目（2003A3080502）；深港创新圈计划资助项目（200701）。