Evaluation of recombinant MGL_1304 produced by Pichia pastoris for clinical application to sweat allergy
Evaluation of recombinant MGL_1304 produced by Pichia pastoris for clinical application to sweat allergy
复制标题
毕赤酵母重组MGL_1304治疗汗液过敏的临床应用评价
DOI：
10.1016/j.alit.2015.03.003
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发表时间：
2015
影响因子：
6.8
通讯作者：
Hide M
中科院分区：
文献类型：
--
作者：
Kan T;Hiragun T;Ishii K;Hiragun M;Yanase Y;Tanaka A;Hide M
背景我们以前发现球形马拉色菌分泌的MGL_1304是特应性皮炎（AD）和胆碱能性荨麻疹（ChU）患者的汗液抗原。然而，从人汗液或M. globosa（sup-MGL_1304）是昂贵且耗时的。此外，使用大肠杆菌表达的重组MGL_1304（TF-rMGL_1304）需要大分子伴侣蛋白，并且缺乏酵母的原始糖基化。因此，我们利用毕赤酵母（Pichia pastoris，P-rMGL_1304）表达了重组MGL_1304蛋白，并对其特性进行了研究。coli和巴斯德毕赤酵母。这些重组抗原和天然抗原的性质进行了比较，通过蛋白质印迹分析，组胺释放试验（HRT）的患者与AD和ChU，和b-氨基己糖苷酶释放试验与RBL-48细胞。结果AD患者血清中P-rMGL_1304特异性IgE与所有MGL_1304重组蛋白及天然抗原均结合。P-rMGL_1304的组胺释放能力比TF-rMGL_1304高100倍，与sup-MGL_1304相当。用AD患者血清致敏的RBL-48细胞对P-rMGL_1304的刺激的脱颗粒率与sup-MGL_1304相当，而TF-rMGL_1304的脱颗粒率相对较弱。结论P-rMGL_1304具有与天然抗原相当的抗原性，在HRT和RBL-48细胞脱颗粒试验中优于TF-rMGL_1304。Copyright© 2015，日本变态反应学会.制作和托管由爱思唯尔BV这是一个开放获取的文章下，CC BY-NC-ND许可证（http：//creativecommons. org/licenses/by-nc-nd/4.0/）
BackgroundWe previously identified MGL_1304 secreted by Malassezia globosa as a sweat antigen for patients with atopic dermatitis (AD) and cholinergic urticaria (ChU). However, purifying native MGL_1304 from human sweat or culture supernatant of M. globosa (sup-MGL_1304) is costly and timeconsuming. Moreover, recombinant MGL_1304 expressed by using Escherichia coli (TF-rMGL_1304) needs a large chaperon protein and lacks the original glycosylation of yeasts. Thus, we generated a recombinant MGL_1304 by Pichia pastoris (P-rMGL_1304) and investigated its characteristic features.MethodsRecombinant MGL_1304 proteins expressed by E. coli and P. pastoris were generated. Properties of these recombinants and native antigens were compared by western blot analysis, histamine release tests (HRT) of patients with AD and ChU, and b-hexosaminidase release tests with RBL-48 cells. P-rMGL_1304-specific IgE in sera of patients with AD were measured by sandwich ELISA.ResultsWestern blot analysis revealed that IgE of patients with AD bound to all MGL_1304 recombinants and native antigens. The histamine releasing ability of P-rMGL_1304 was 100 times higher than that of TF-rMGL_1304, and was comparable to that of sup-MGL_1304. Degranulation rates of RBL-48 cells, sensitized with sera of patients with AD in response to the stimulation of P-rMGL_1304, were comparable to those of sup-MGL_1304, whereas those of TF-rMGL_1304 were relatively weak. The levels of P-rMGL_1304-specific IgE in sera of patients with AD were correlated with their disease severities.ConclusionsP-rMGL_1304 has an antigenicity comparable to the native antigen, and is more useful than TF-rMGL_1304, especially in HRT and degranulation assay of RBL-48 cells. Copyright© 2015, Japanese Society of Allergology. Production and hosting by Elsevier BV This is an open access article under the CC BY-NC-ND license (http://creativecommons. org/licenses/by-nc-nd/4.0/)