抗黄曲霉毒素合成中关键蛋白AFLP(OmtA)的单克隆抗体制备与鉴定

摘要

AFLP(OmtA) protein is a key enzyme in the late period of aflatoxin synthesis.SUMO-AFLP fusion protein expressed by Escherichia coli was used as immunogen to immunize Balb/c mice.Spleen cells of positive serum mice were selected and fused with SP2/0 myeloma cells.Monoclonal hybridoma cells were screened by indirect ELISA and limited dilution.Thus the hybridoma cell lines were screened with stable secretion of anti-AFLP monoclonal antibody.Ascites antibodies were induced and prepared by the positive cell line,and were purified by caprylic acid / ammonium sulfate precipitation.By indirect ELISA and Western blotting,one hybridoma cell line 3B4 3B10 was found which could secret the anti-AFLP monoclonal antibody,and the subtype was IgG2a,The antibody ascites iter was 1∶256 000.Western blotting showed that the anti-AFLP monoclonal antibody specifically recognized both recombinant and native AFLP proteins in Aspergillus flavus.By 1 μg · mL-1 monoclonal antibody detection,the linear range was 1.259-57.335ng · mL-1 with the limit of 0.851 ng · mL-1 (R2 =0.998 3).In conclusion,monoclonal antibody against native AFLP protein was successfully prepared with high specificity and sensitivity,which laid foundation for further study in AFLP expression and functional mechanism.%AFLP (OmtA)蛋白是黄曲霉毒素合成晚期的关键酶.用大肠杆菌表达的重组SUMO-AFLP融合蛋白作为免疫原,免疫Balb/c小鼠,选取小鼠阳性血清的脾细胞与SP2/0骨髓瘤细胞融合,用间接ELISA和有限稀释法进行单克隆杂交瘤细胞的筛选,筛选出可以稳定分泌抗AFLP单克隆抗体的杂交瘤细胞株.再用阳性细胞株制备腹水抗体,经辛酸-硫酸铵纯化抗体后,用间接ELISA、Western杂交等方法对抗体的特性进行鉴定.筛选出1株能稳定分泌抗AFLP单克隆抗体的细胞株3B4 3B10,亚类鉴定单抗为IgG2a.间接ELISA法测定该细胞腹水抗体的效价为1∶256 000,Westem杂交结果显示抗AFLP单克隆抗体能特异性识别AFLP重组蛋白及黄曲霉菌内的天然AFLP蛋白.用1μg·mL-1单克隆抗体检测AFLP蛋白,其线性检测范围为1.259 ～ 57.335ng· mL-1,检测限为0.851ng·mL-1,R2 =0.998 3.本文成功制备抗黄曲霉毒素合成中天然AFLP蛋白的单克隆抗体,特异性和灵敏度均较高,为进一步研究AFLP的表达及功能机制奠定了基础.