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以优异小麦品种“小偃6号”的高分子量麦谷蛋白亚基 (high molecular weight glutenin subunit.HMW—GS)1Bxl4和lBvl5为混合抗原,免疫BALB/c小鼠.将其脾细胞和骨髓瘤细胞(SP2/0)融合,采用间接ELISA 筛选与有限稀释法克隆,建立了一株稳定分泌单克隆抗体的杂交瘤细胞系.单抗Ig亚类为IgGl.蛋白质免疫印迹实验结果表明:该单抗能与小麦所有HMW-GS发生强烈反应。而与醇溶蛋白和低分子量谷蛋白亚基不反应.能与普通小麦近缘种粗山羊草、硬粒小麦、黑麦和大麦的高分子量贮藏蛋白发生反应:而与燕麦、玉米、高粱、谷子和水稻等禾谷类作物贮藏蛋白不发生反应.另外,利用原核表达载体pGEX-4T-1,将HMw—GSN 端、中央重复区和C 端3个结构域分别在E.coli BL2l中融合表达.免疫印迹实验结果表明,该单抗识别的抗原决定簇应位于中央重复区中的六肽和九肽之中.对该单抗的抗原决定簇及其在小麦品质育种中的运用进行了讨论.
BALB / c mice were immunized with mixed antigen of high molecular weight glutenin subunit (HMMW-GS) 1Bxl4 and lBvl5 of excellent wheat cultivar “Xiaoyan 6.” The spleen cells and myeloma cells (SP2 / 0), and a monoclonal antibody-secreting hybridoma cell line was established by indirect ELISA screening and limiting dilution method.The monoclonal antibody Ig subclass was IgG1.The results of Western blotting showed that the monoclonal antibody Strongly reactive with all HMW-GS in wheat. But did not react with gliadin and low molecular weight glutenin subunits, and could react with the high molecular weight storage proteins of common wheat relatives Aegilops such as Tilapia, Triticale, Rye and Barley, while with oats, maize, sorghum, Millet and rice and other cereal crops storage protein did not react.In addition, the prokaryotic expression vector pGEX-4T-1, HMw-GSN end, the central repeat and C-terminal three domains were fused in E. coli BL21 expression The results of Western blotting showed that the epitopes identified by this McAb should be located in the hexapeptide and nonapeptide in the central repeat region.The antigenic determinants of this McAb and its application in wheat quality breeding were discussed .