抗体夹心BGT-ELISA方法的建立及在转基因白桦中的应用  

Construction and Application of BGT-ELISA on Transgenic Birch

作　　者：曾凡锁[1] 骆薇[1] 赵宏翠[1] 詹亚光[1] 孙东[1] 

机构地区：[1]东北林业大学生命科学学院林木遗传育种与生物技术教育部重点实验室,哈尔滨150040

出　　处：《中国农学通报》2010年第1期67-73,共7页Chinese Agricultural Science Bulletin

基　　金：国家自然科学基金项目(30872045);教育部博士点新教师基金(200802251038)

摘　　要：以蜘蛛杀虫肽与Bt-toxinC肽融合蛋白基因(bgt基因)的表达产物的定量分析为研究目标,采用原核表达及纯化出融合蛋白His-BGT作为抗原进行兔免疫,得到相应的抗血清,采用ELISA法检测效价在1:10000以上,并对抗血清进行了免疫亲和层析,获得了高纯度的IgG,Westernblot检测具有较好的特异性。采用过碘酸钠法将抗体标记辣根过氧化物酶(HRP),得到第二抗体即酶标抗体,标记率在85%以上。建立起检测BGT杀虫蛋白的快速、灵敏的方法。应用该检测方法,分析了不同转基因白桦(BetulaplatyphyllaSuk.)株系中BGT蛋白含量占叶片可溶性总蛋白含量的0.05%～0.3%,并利用Westernblot验证了此方法是可靠的。说明抗体夹心BGT-ELISA方法能够定量分析转基因植株中BGT蛋白的含量,为转bgt基因植物的检测和应用奠定了基础。This paper took expressed product of BGT gene consisting of the insecticidal toxin gene from the spider, Atrax robustus, and the C terminal of Cry IA （b） gene from Bacillus thuringiensis in transgenic birch as research target. Through IPTG induction and metal chelate affinity chromatography purifica- tion, fusion protein His-BGT with high purity and its specific antibody have been obtained. The fusion protein as antigen was used to immunize rabbit for the preparation of polyclonal antibodies with high ti- ter. The titer of serum antibody was above 1：10 000 as detected by ELISA and purified by immuno-af- finity chromatography. The high purity of IgG was obtained. Western blot assay demonstrated that the IgG fusion protein after purification showed definite specificity. With the method of NaIO4 anti-BGT antibody was labeled with HRP to make enzyme-labeled antibody. The labeling rate was above 85%. An ELISA bi-antibody sandwich for the detection of BGT protein in transgenie birch has been established. The method has been preliminary optimized. A rapid and sensitive method for detecting BGT insecticid- al protein was preliminary established. The BGT expression level of 5 month in the plants of BGT transgenie birch ranged from 0.05% to 0.3% of total soluble protein by established detection method. And this method was reliable that was validated by western blot.

关 键 词：转基因白桦 多克隆抗体 酶标抗体 酶联免疫 

分 类 号：S718.3[农业科学—林学]