论文部分内容阅读
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。
The purpose of this study was to establish a set of suitable screening methods for identifying the type of lipoxygenase deficiency in breeding progenies. Firstly, we selected the subtractive immunization method which is suitable for the near-isogenic lineage material. Using J12-2 with single Lox2 as tolerance, Soybean Lox (Type IB) containing three Loxs as antigen, Lox2-1, a hybridoma cell line that secretes Lox2 monoclonal antibodies. On the basis of solving the Lox2 monoclonal antibody, the purified Lox1 and Lox3 were further used as antigen to prepare and screen a hybridoma cell line Lox1-1 and Lox3 which can stably secrete Lox1 monoclonal antibody according to a conventional method Tumor cell lines Lox3-1, Lox3-2, Lox3-3 and Lox3-4; ascites induced cell line antibody production, ascites titer between 1:20 000 and 1: 800 000, identified six single Anti-all have strong specificity. Rabbit polyclonal antibody sera were prepared using Soybean Lox (TypeI-B) as an antigen and their titer against Lox1, Lox2 and Lox3 reached 1:76 800. Finally, a technology system for identifying the type of soybean lipoxygenase deficiency by using rabbit polyclonal antibody as the solid phase antibody, Lox1-1, Lox2-1 and Lox3-4 as the detection antibody was finally established. This technique not only provided a non-bloody soybean breeding Provide technical support, but also for other isozyme detection technology has accumulated valuable experience.