大豆脂肪氧化酶同功酶联合鉴定技术的建立  

Establishment of A Joint Identification Technology of Soybean Lipoxygenases

作　　者：马志民[1] 秦君[1] 杨春燕[1] 邸锐[1] 闫龙[1] 张梅申[2] 李子千[1] 李亚璞[3] 史晓蕾[1] 鲍聪 许宁 封树平[1] 张孟臣[1] 

机构地区：[1]河北省农林科学院粮油作物研究所,国家大豆改良中心石家庄分中心,农业部黄淮海大豆生物学与遗传育种重点实验室,河北省遗传育种重点实验室,河北石家庄050035 [2]河北省农林科学院,河北石家庄050000 [3]河北省科学院生物研究所,河北石家庄050081 [4]河北省农业技术推广总站,河北石家庄050011

出　　处：《河北农业科学》2014年第2期52-59,共8页Journal of Hebei Agricultural Sciences

基　　金：国家"863"计划项目(2012AA101106);国家现代农业产业技术体系建设专项(CARS-004-PS06);十二五国家科技支撑计划项目(2011BAD35B06);河北省自然科学重点基金项目(C2012301020)

摘　　要：本研究目的是建立一套适宜于育种后代材料脂肪氧化酶缺失类型筛选鉴定的方法。首先选择适用于近等基因系材料的消减免疫法,以单缺Lox2的J12-2作为耐受原,将含有3种Loxs的Soybean Lox(Type I-B)作为抗原,利用该方法筛选到1株能稳定分泌Lox2单抗的杂交瘤细胞株Lox2-1。在解决了Lox2单抗的基础上,进一步以纯化的Lox1和Lox3作抗原,按常规方法,制备并筛选到1株能稳定分泌Lox1单抗的杂交瘤细胞株Lox1-1,4株Lox3的杂交瘤细胞株Lox3-1、Lox3-2、Lox3-3和Lox3-4;利用腹水诱导法生产其细胞株抗体,腹水效价在1∶20 000与1∶800 000之间,经鉴定6株单抗均具有较强的特异性。以Soybean Lox(TypeI-B)作抗原制备兔多克隆抗体血清,其针对Lox1、Lox2和Lox3的效价均达到1∶76 800。最终建立了以兔多抗作固相抗体,Lox1-1、Lox2-1、Lox3-4分别作检测抗体的对大豆脂氧酶缺失类型进行联合鉴定的技术体系,该技术不但为无腥大豆育种提供了技术保障,还为其他同工酶检测技术的开发积累了宝贵经验。It was established in this study that applies to the missing type screening and identification of the soybean lipoxygenases for the breeding of the non-odor soybean.Selection the method which suitable for near isogenic lines of subtractive immunization, and using J12-2 missing Lox2 as the toleragen and Soybean Lox （Type I-B） containing Lox1 , Lox2 and Lox3 simultaneously as the antigen, and hybridomas secreting antibodies against Lox2 were selected and cloned.In the settlement of the foundation of Lox2 monoclonal antibody, the hybridoma cell lines secreting anti-Lox1 and anti-Lox3 McAbs respectively were obtained by immunizing the BALB/C mice with the soybean lipoxygenases purified by ourselves, and one stable McAbs Lox1-1 against Lox1, and four （ Lox3-1, Lox3-2, Lox3-3, Lox3-4 ） against Lox3.These hybridomas were prepared ascites of the mice, and the titers of the McAbs in the ascites ranged from 1∶20 000 to 1∶800 000, and these McAbs all had strong specificity.The rabbit polyclonal antibody in serum anti Lox1 , Lox2 and Lox3 simultaneously was obtained by immunizing the New Zealand white rabbits with Soybean Lox （ Type I-B） , and the titer all achieves 1∶76 800 that against Lox1, Lox2 and Lox3 respectively.In the end, the technical system for the the missing type screening and identification of the soybean lipoxygenases was established using the rabbit polyclonal antibody as the solid-phase antibody, and the McAbs Lox1-1, Lox2-1, Lox3-4 as the detectable antibodies respectively.It provided the technical support for the breeding of the non-odor soybean and accumulating valuable experience for developing the other isoenzyme detection technology.

关 键 词：脂肪氧化酶 同功酶 单克隆抗体 多克隆抗体 双抗体夹心法 联合鉴定 

分 类 号：S565.1[农业科学—作物学]