大豆脂肪氧化同工酶(Lox1)单克隆抗体的制备

利用薄层等电聚焦电泳检测缺失大豆脂氧酶近等基因系-Su系列,将标记不同缺失类型种子经脱脂、干燥、浸提、离心后制成样品,通过非变性聚丙烯酰胺凝胶电泳分离和电泳洗脱仪回收获得蛋白。间接ELISA测定Lox1免疫血清效价为1∶1600,免疫BALB/C小鼠的脾细胞和达到对数生长期的SP2/0骨髓瘤细胞融合培养,筛选杂交瘤细胞的阳性克隆,应用有限稀释法和交叉分析,获得3株能稳定传代且分泌Lox1单克隆抗体的、与其他Lox类型没有交叉反应的杂交瘤细胞,各株单抗的效价均在1∶64以上。大豆脂肪氧化同工酶Lox1单克隆抗体用于检测脂氧酶的缺失类型,可为辅助育种提供投资少、见效快的鉴定方法,可为研制大豆脂肪氧化同工酶检测试剂盒提供实验基础。

Preparation of Monoclonal Antibodies to Soybean Lipoxygenase( Lox1 )

Isoelectric focusing in thin-layer polyacrylamide gels(IEF-PAGE) were applied to analyze lipoxygenase lacking types near iosogenic line(NIL) of soybeans.Seeds with marked types were managed by degreasing,centrifugating and drying with vacuumized.Three lipoxygenase isozymes were separated clearly from crude seed extracts by native polyacrylamide gel eletrophoretic technique,and three protein bands were eluted and purified right.Considered activity and purity of three lipoxygenases,Lipoxygenase-1 can be regarded as appropriate antigen.The minimum amount of antiserum could successfully detect 10 ug/ml antigen with 1:1600 dilution of antiserum by indirect enzyme-linked immunosorbent assay(ELISA).Hybridoma cell lines of secreting monoclonal antibodies(MAbs) were fused with myeloma cells(SP2/0) and spleen cells from BALB/C mouse,which immunized by Lox1 particles.Three MAbs present positive through limited diluting and cross-reacting by Indirect ELISA,and do not cross-react with other tested soybean lipoxygenase proteins.Then,monoclonal antibodies using hybridoma fusion can identify isozyme to be present or absent,and this test results can provide an efficient and cheap determining method to assist breeding and experiment base in order to develop reagent box.