Molecular cloning and expression in insect cells of honeybee venom allergen acid phosphatase (Api m 3)
Molecular cloning and expression in insect cells of honeybee venom allergen acid phosphatase (Api m 3)
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DOI：
10.1016/j.jaci.2005.12.1331
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发表时间：
2006-04-01
影响因子：
14.2
通讯作者：
Ollert, MW
中科院分区：
文献类型：
--
作者：
Grunwald, T;Bockisch, B;Ollert, MW
背景资料：酸性磷酸酶（API m 3）是蜜蜂（Apis mellifera）毒液中的一种主要变应原，其重组蛋白的获得将有助于改进诊断试验和免疫治疗的发展。目的：确定API m 3的完整一级结构，并在昆虫细胞中表达的基础上获得重组API m 3。方法：克隆API no 3前毒腺cDNA，并在真核昆虫细胞中表达。结果：PCR扩增得到一个1122bp的DNA片段，经多种方法鉴定其为API的编码序列。在粉纹夜蛾细胞中表达的重组API m 3在pH 4.5下显示出预期的分子量和酶活性。通过质谱分析纯化的重组API m 3的胰蛋白酶片段证实了其身份。在免疫分析中，重组API m 3特异性识别的IgE抗体的混合血清在Western印迹和37%的个体血清的蜂毒致敏patients在ELISA analysis.Conclusion：重组API m 3的可用性提供了一种工具，为改进的诊断试验的发展和设计更安全，更有效的免疫学方法蜂毒过敏。临床含义：重组蜂毒过敏原API m 3是寻找蜜蜂毒液过敏的最佳组分解析方法的关键要素，该方法涉及上级诊断测试的开发和过敏原免疫治疗的改进。
Background: Acid phosphatase (Api m 3) is a major allergen in honeybee (Apis mellifera) venom, and its availability as a recombinant protein may facilitate the development of improved diagnostic tests and immunotherapies.Objective: One objective is the determination of the complete primary structure of Api m 3 and to obtain recombinant Api m 3 on the basis of expression in insect cells. Another objective is the quantitative analysis of patient serum IgE antibody reactive to recombinant Api m 3.Methods: The cloning of Api no 3 front venom gland cDNA and its expression as a full-length protein in eukaryotic insect cells is described. The immunoreactivity of serum IgE antibodies of honeybee venom-sensitized patients to recombinant Api m 3 was determined in an enzyme immunoassay.Results: PCR amplification generated a 1122-bp DNA fragment whose identity as the coding sequence of Api m 3 was verified by several means. Recombinant Api m 3, expressed in Trichoplusia ni cells, showed an expected molecular weight and enzymatic activity at pH 4.5. Analysis of tryptic fragments of purified recombinant Api m 3 by mass spectrometry confirmed its identity. In immunoassays, recombinant Api ni 3 is specifically recognized by IgE antibodies of pooled serum in Western blots and by 37% of the individual sera of honeybee venom-sensitized patients in ELISA analysis.Conclusion: The availability of recombinant Api m 3 provides a tool for both the development of improved diagnostic tests and the design of safer and more effective immunotherapeutic approaches for honeybee venom allergy. Clinical implications: The recombinant venom allergen Api m 3 is a key element in the search for an optimized component resolved approach to honeybee venom allergy with regard to both the development of superior diagnostic tests and the improvement of allergen immunotherapy.