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Production and evaluation of recombinant allergens with carbohydrate epitopes expressed in insect cells

具有在昆虫细胞中表达的碳水化合物表位的重组过敏原的生产和评估

基本信息

批准号：
12556060
负责人：
MATSUDA Tsukasa
金额：
$ 4.86万
依托单位：
NAGOYA UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2001
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-12556060/
关键词：
Allergy Carbohydrate antigen Insect cells Plant allergens

项目摘要

Several glycoproteins with asparagine-linked sugar chains were expressed in insect cells, and the glycoproteins with fucose-containing sugar chains were produced. First, the cDNA encoding each protein was inserted into a transfer vector to introduce into baculovirus genome DNA. The viral genome DNA and the transfer vector containing the CDNA were introduced into a insect cell line, BTI TN 5B1-4. The virus particles were recovered from the culture supernatant, and its betagalactosidase activity was measured. The galactosidase-positive recombinant virus was selected and cloned. Each recombinant virus was cultured in a larger scale, and recombinant glycoproteins expressed in the insect cells were purified. To determine whether sugar chains with the carbohydrate epitopes were added to the recombinant proteins, the purified glycoproteins were subjected to immunoblotting and ELISA using the antiserum specific for the carbohydrate epitope. Both of the blot and ELISA demonstrated that all the recombinant glycoproteins tested were clearly positive to the carbohydrate epitope-specific antibody, though the reactivity varied from one protein and another. Furthermore, the presence of the carbohydrate epitope was confirmed from the observation that the antigenic reactivity of each recombinant glycoprotein was lost by the periodate treatment. Further detailed analyzes to characterize the carbohydrate structure are in progress including lectin-binding assay, glycosidase treatments, chemical analysis, and immunochemical analyzes using patient's antibodies. The present research demonstrated that recombinant glycoprotein allergens with carbohydrate epitopes can be produced in the baculovirus/insect cell expression system, and suggests that the recombinant proteins show allergenic reactivity comparable to natural allergens. Thus, baculovirus/insect cell expression system would be a useful tool for theproduction of glycoprotein allergens for research and diagnosis purposes.

在昆虫细胞中表达了几种含天冬酰胺糖链的糖蛋白，并产生了含岩藻糖糖链的糖蛋白。首先，将编码每种蛋白质的cDNA插入转移载体中以引入杆状病毒基因组DNA中。将病毒基因组DNA和含有cDNA的转移载体引入昆虫细胞系BTI TN 5 B1 -4中。从培养上清液中回收病毒颗粒，并测定其β-半乳糖苷酶活性。选择半乳糖苷酶阳性的重组病毒并进行克隆。将每种重组病毒以较大规模培养，并纯化在昆虫细胞中表达的重组糖蛋白。为了确定是否将具有碳水化合物表位的糖链添加到重组蛋白中，使用对碳水化合物表位特异性的抗血清对纯化的糖蛋白进行免疫印迹和ELISA。免疫印迹和ELISA结果均表明，所有重组糖蛋白对糖表位特异性抗体均呈阳性反应，但不同蛋白的反应性不同。此外，碳水化合物表位的存在从观察到的每种重组糖蛋白的抗原反应性通过高碘酸盐处理而丧失而得到证实。进一步详细的分析，以表征碳水化合物的结构正在进行中，包括凝集素结合测定，糖苷酶治疗，化学分析，并使用患者的抗体免疫化学分析。目前的研究表明，重组糖蛋白过敏原与糖抗原表位可以在杆状病毒/昆虫细胞表达系统中产生，并表明重组蛋白显示过敏反应与天然过敏原。因此，杆状病毒/昆虫细胞表达系统将是一个有用的工具，生产糖蛋白过敏原的研究和诊断的目的。