经合理的序列重组制备花生主要过敏原 Ara h 2低致敏衍生物

中国生物工程杂志

2011, Vol. 31

Issue (7): 54-59

研究报告

经合理的序列重组制备花生主要过敏原 Ara h 2低致敏衍生物

易海涛^1,2, 刘芳³, 夏立新^2,4, 闫浩^1,2, 刘飞燕^2,4, 李建杰^1,2, 刘志刚^2,4

1. 深圳大学生命科学学院深圳 518060;
2. 深圳大学医学院过敏反应与免疫学研究所深圳 518060;
3. 新疆华世丹药业股份有限公司乌鲁木齐 830000;
4. 深圳大学医学院深圳 518060

Hypoallergenic Derivatives of the Major Peanut Allergen Ara h 2 Obtained by Rational Sequence Reassembly

YI Hai-tao^1,2, LIU Fang³, XIA Li-xin^2,4, YAN Hao^1,2, LIU Fei-yang^2,4, LI Jian-jie^1,2, LIU Zhi-gang^2,4

1. College of Life Science,Shenzhen University, Shenzhen 518060, China;
2. Institute of Allergy and Immunology, Shenzhen University, Shenzhen 518060, China;
3. Xinjiang Huashidan Pharmaceutical Co., Ltd,Wulumuqi 830000,China;
4. Medical College, Shenzhen University, Shenzhen 518060, China

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摘要：

目的:构建经序列重组的Ara h 2表达载体,表达并纯化该蛋白,鉴定其低致敏原性。方法:根据已鉴定的Ara h 2 IgE抗原表位,利用基因工程技术将Ara h 2基因进行合理的组合,并将其序列进行合成,再将合成后的基因连入原核表达载体pET-32a(+)上,然后转入Origami宿主表达菌中;IPTG诱导表达;通过Ni²⁺亲和层析(FPLC)纯化目的蛋白;Western blotting和ELISA鉴定该重组蛋白的低致敏原性。结果:测序结果表明合成后的序列成功转入原核表达载体pET-32a(+)上。重组蛋白纯化后经SDS-PAGE鉴定,目的蛋白大小与理论值相符。Western blotting和ELISA结果均表明通过基因工程改造的Ara h 2蛋白(S-Ara h 2)与重组的Ara h 2(R-Ara h 2)蛋白相比,结合花生过敏病人混合血清中IgE显著降低。结论:成功构建了经序列重组的Ara h 2表达载体,该基因表达的重组蛋白具有良好的低致敏原性,这将会为花生过敏患者的脱敏治疗提供了新的安全疫苗基础。

关键词： 花生; 重组; 过敏原Ara h 2; 低致敏衍生物; 疫苗

Abstract:

Objective: To express and purify the peanut major allergen Ara h 2 that obtained by rational sequence reassembly and preliminarily characterize the hypoallergenic of purified recombinant S-Ara h 2 protein. Methods: The Ara h 2 sequence was reassembled and inserted into the expression vector pET-32a(+). The vector was transformed into Origami and the protein expression was induced by IPTG. Ni²⁺chelating affinity chromatography was used to purify the recombinant S-Ara h 2 protein. The hypoallergenic of S-Ara h 2 was examined by Western blotting and ELISA. Results: The ORF which contained 471 bp and encoded 157 amino acids was authenticated to be S-Ara h 2. The recombinant S-Ara h 2 protein,induced by IPTG,which is consistent with the actual value. The affinity between recombinant S-Ara h 2 protein and IgE antibodies from pooled peanut-allergic patients serum was significant decrease compared with R-Ara h 2 was identified by Western blotting and ELISA. Conclusion: Recombinant S-Ara h 2 protein was obtained with hypoallergenic. It will provide a novel safe vaccine to cure peanut allergy patients by Allergen-specific immunotherapy.

Key words: Arachis hypogaea L. Recombination Allergen Ara h 2 Hypoallergenic derivatives Vaccine

收稿日期: 2011-03-31 出版日期: 2011-07-25

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引用本文:

易海涛, 刘芳, 夏立新, 闫浩, 刘飞燕, 李建杰, 刘志刚. 经合理的序列重组制备花生主要过敏原 Ara h 2低致敏衍生物[J]. 中国生物工程杂志, 2011, 31(7): 54-59.

YI Hai-tao, LIU Fang, XIA Li-xin, YAN Hao, LIU Fei-yang, LI Jian-jie, LIU Zhi-gang. Hypoallergenic Derivatives of the Major Peanut Allergen Ara h 2 Obtained by Rational Sequence Reassembly. China Biotechnology, 2011, 31(7): 54-59.

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https://manu60.magtech.com.cn/biotech/CN/ 或 https://manu60.magtech.com.cn/biotech/CN/Y2011/V31/I7/54

[1] Nicolaou N, Poorafshar M, Murray C, et al. Allergy or tolerance in children sensitized to peanut: prevalence and differentiation using component-resolved diagnostics. J Allergy Clin Immunol, 2010,125(1):191-197.

[2] 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 Clin Immunol, 2003,112(6):1203-1207.

[3] Bock S A, Munoz-Furlong A, Sampson H A. Fatalities due to anaphylactic reactions to foods. J Allergy Clin Immunol,2001,107(1):191-193.

[4] Bock S A, Munoz-Furlong A, Sampson H A. Further fatalities caused by anaphylactic reactions to food, 2001-2006. J Allergy Clin Immunol,2007,119(4):1016-1018.

[5] 王通,梁炫强,李玲.花生致敏原的研究进展.中国油料作物学报,2007,29(3):353-358. Wang T, Liang X Q, Li L. Chinese Journal of Oil Crop Sciences, 2007,29(3):353-358.

[6] Fleischer D M. The natural history of peanut and tree nut allergy. Curr Allergy Asthma Rep, 2007,7(3):175-181.

[7] Varshney P, Jones S M, Scurlock A M,et al. A randomized controlled study of peanut oral immunotherapy: Clinical desensitization and modulation of the allergic response. J Allergy Clin Immunol,2011, 127(3):654-660.

[8] Pieretti M M, Chung D, Pacenza R,et al. Audit of manufactured products: use of allergen advisory labels and identification of labeling ambiguities.J Allergy Clin Immunol, 2009,124(2):337-341.

[9] 张田勘. 花生过敏:小问题大麻烦. 知识就是力量,2008,11(11):22-24. Zhang T K. Knowledge is Power,2008,11(11):22-24.

[10] Thalhamer T, Dobias H, Stepanoska T,et al. Designing hypoallergenic derivatives for allergy treatment by means of in silico mutation and screening. J Allergy Clin Immunol, 2010,125(4):926-934.

[11] Bousquet J, Lockey R, Malling H J, et al. Allergen immunotherapy: therapeutic vaccines for allergic diseases. World Health Organization. American academy of Allergy, Asthma and Immunology. Ann Allergy Asthma Immunol,1998,81(5):401-405.

[12] Valenta R. The future of antigen-specific immunotherapy of allergy. Nat Rev Immunol, 2002,2(6):446-453.

[13] Ferreira F, Wallner M, Thalhamer J. Customized antigens for desensitizing allergic patients. Adv Immunol,2004,84:79-129.

[14] Jutel M, Jaeger L, Suck R, et al. Allergen-specific immunotherapy with recombinant grass pollen allergens. J Allergy Clin Immunol,2005,116(3):608-613.

[15] Hefle S L, Furlong T J, Niemann L, et al. Consumer attitudes and risks associated with packaged foods having advisory labeling regarding the presence of peanuts. J Allergy Clin Immunol, 2007,120(1) :171-176.

[16] Larche M, Akdis C A, Valenta R. Immunological mechanisms of allergen-specific immunotherapy.Nat Rev Immunol,2006,6(1):761-771.

[17] Oppenheimer J J, Nelson H S, Bock S A, et al. Treatment of peanut allergy with rush immunotherapy. J Allergy Clin Immunol, 1992,90(2):256-262.

[18] Stanley J S, King N, Burks A W,et al. Identification and mutational analysis of the immunodominant IgE binding epitopes of the major peanut allergen Ara h 2. Arch Biochem Biophys, 1997,342(2):244-253.

[19] 夏立新,闫浩,汤慕瑾,等. 花生过敏原Ara h2与Ara h6的生物信息学比较研究. 深圳大学学报(理工版),2010,27(2):241-245. Xia L X, Yan H, Tang M J, et al. Journal of Shenzhen University Science and Engineering,2010,27(2):241-245.

[20] Brown S J, Asai Y, Cordell H J,et al.Loss-of-function variants in the filaggrin gene are a significant risk factor for peanut allergy. J Allergy Clin Immunol, 2011,127(3):661-667.

[21] Valenta R. Recombinant allergens.Allergy, 1998,53(6):552-556.

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