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  

Preparation and identification of monoclonal antibodies against AFLP(OmtA),a key protein involved in aflatoxin biosynthesis

作　　者：王婷[1,2,3] 李培武[1,2,3,4,5] 张奇[1,2,3] 张文[1,3,5] 张兆威[1,2,4] 

机构地区：[1]中国农业科学院油料作物研究所,湖北武汉430062 [2]农业部油料作物生物学与遗传育种重点实验室,湖北武汉430062 [3]农业部生物毒素检测重点实验室,湖北武汉430062 [4]农业部油料产品质量安全风险评估实验室,湖北武汉430062 [5]农业部油料及制品质量监督检验测试中心,湖北武汉430062

出　　处：《中国油料作物学报》2017年第4期551-555,共5页Chinese Journal of Oil Crop Sciences

基　　金：公益性农业行业专项(201203094;201203037);中国农业科学院院所长基金(1610172010003)

摘　　要：AFLP(Omt A)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Ig G2a。间接ELISA法测定该细胞腹水抗体的效价为1∶256 000,Western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·m L^(-1)单克隆抗体检测AFLP蛋白,其线性检测范围为1.259~57.335ng·m L^(-1),检测限为0.851ng·m L^(-1),R~=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。AFLP（Omt A） 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 line3B4 3B10 was found which could secret the anti-AFLP monoclonal antibody,and the subtype was Ig G2 a. 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 1μg·m L-1monoclonal antibody detection,the linear range was 1. 259-57. 335ng·m L-1with the limit of 0. 851 ng·m L-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.

关 键 词：黄曲霉毒素 AFLP 单克隆抗体 制备与鉴定 

分 类 号：S379.7[农业科学—农产品加工]