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

在昆虫细胞中表达了几种具有天冬酰胺连接糖链的糖蛋白，并产生了含焦糖链的糖蛋白。首先，将编码每个蛋白的cDNA插入转移载体，导入杆状病毒基因组DNA。将病毒基因组DNA和含有CDNA的转移载体导入昆虫细胞系BTI TN 5B1-4。从培养上清中回收病毒颗粒，测定其β -半乳糖苷酶活性。选择并克隆了半乳糖苷酶阳性重组病毒。对重组病毒进行了大规模培养，并纯化了在昆虫细胞中表达的重组糖蛋白。为了确定重组蛋白中是否添加了含有碳水化合物表位的糖链，将纯化的糖蛋白用碳水化合物表位特异性抗血清进行免疫印迹和ELISA检测。免疫印迹和酶联免疫吸附试验表明，所有重组糖蛋白对碳水化合物表位特异性抗体均明显阳性，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中得到证实，每个重组糖蛋白的抗原反应性都因高碘酸盐处理而丧失。进一步详细分析表征碳水化合物结构的工作正在进行中，包括凝集素结合试验、糖苷酶处理、化学分析和使用患者抗体的免疫化学分析。本研究表明，具有碳水化合物表位的重组糖蛋白过敏原可以在杆状病毒/昆虫细胞表达系统中产生，并表明重组蛋白具有与天然过敏原相当的致敏性。因此，杆状病毒/昆虫细胞表达系统将为糖蛋白过敏原的研究和诊断提供一个有用的工具。

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.