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

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.