抗曲霉硫氧还蛋白还原酶单克隆抗体的制备及鉴定

Preparation and identification of monoclonal antibody against Aspergillus thioredoxin reductase GliT

目的制备烟曲霉硫氧还蛋白还原酶(thioredoxin reductase,TR)GliT单克隆抗体(单抗)并进行鉴定。方法取重组烟曲霉TR蛋白免疫的BALB/c小鼠脾细胞与骨髓瘤细胞Sp2/0融合,筛选分泌抗TR抗体的杂交瘤细胞,有限稀释法克隆化。用ELISA法测定抗体效价,IsoStrip鉴定抗体类、型,用western blot、免疫荧光分析技术和免疫组化染色鉴定单抗的特异性。结果筛选到1株稳定分泌抗TR单抗(Anti-TR1)的杂交瘤细胞,抗体亚类为IgG1,轻链为κ型。ELISA法测定效价为5×105,能够与烟曲霉分泌蛋白及菌丝中天然TR特异性结合,还可与基因重组TR特异性结合。免疫荧光检测表明,该株单抗与烟曲霉、黄曲霉和黑曲霉菌丝均发生反应,是曲霉属特异性的抗体。病理组织免疫组化染色结果显示,该抗体可与曲霉菌丝产生特异性反应。结论获得了1株持续分泌高效价抗曲霉属特异性抗TR单抗的交杂瘤细胞株,为进一步的研究奠定了基础。

Objective To prepare the monoclonal antibody against Aspergillusfumigatus thioredoxin reductase GliT （TR）, and identify its properties. Methods The spleen cells from BALB/c mice immunized with the recombinant TR were hybridized with myeloma cell Sp2/0. Then, the hybridized cells secreting antibodies against TR were selected for cloning by limiting dilution assay. The titer and specificity of the monoclonal antibodies were determined by ELISA, western blot, immunofluorescence assay and immunohistochemisty, respectively, and the class and type of the antibodies were identified by IsoStrip reagent. Results A hybridized cell clone stably se- creting anti-TR monoclonal antibody （ named Anti-TR1 ） was selected. The subclass of Anti-TR1 was IgG1 and its light chain belonged to K-type. ELISA showed that the titer of Anti-TR1 was 5 × 105. western blot showed that Anti-TR1 could specifically bind with natural and recombinant TR. Immunofluorescence assay and immunohistochemisty indicated that Anti-TR1 could react with the hypha of Asper- gillus Spp. Conclusion A hybridoma cell line which could continuously secret the monoclonal antibody against TR of Aspergillus Spp with high titer and specificity is successfully obtained, which provides some basis for the further study of TR.