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中国农学通报

中国农学通报 ›› 2009, Vol. 25 ›› Issue (5): 232-235.

所属专题： 生物技术； 烟草种植与生产

• 植物保护 农药 • 上一篇    下一篇

双抗夹心ELISA方法检测转cry8Ca基因烟草的杀虫蛋白

王容燕,,董建臻,王金耀,郎志宏,曹伟平,宋福平,杜立新,宋 健,冯书亮   

  • 收稿日期:2008-12-01 修回日期:2009-01-16 出版日期:2009-03-05 发布日期:2009-03-05

Detection of insecticidal protein in transgenic cry8Ca tobacco

Wang Rongyan,, Dong Jianzhen, Wang Jinyao, Lang Zhihong, Cao Weiping, Song Fuping, Du Lixin, Song Jian, Feng Shuliang   

  • Received:2008-12-01 Revised:2009-01-16 Online:2009-03-05 Published:2009-03-05

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摘要： 为了检测转cry8Ca基因烟草的杀虫蛋白，建立了双抗夹心ELISA检测体系。确定多克隆抗体浓度为1:3200，酶标结合物浓度为1:400，pH 9.6碳酸缓冲液提取，pH7.4磷酸缓冲液包被，0.5%明胶-PBST封闭，TMB底物作用15min为最佳双抗夹心ELISA检测条件。采用这一检测体系，对转cry8Ca基因烟草植株进行检测，S12株系中的植株Cry8C蛋白含量最高，每克鲜重含有12.683-14.461ug，占总蛋白量的0.052%-0.062%。

关键词: 现代, 现代, 后现代, 农村教育, 特征

Abstract: The reaction conditions for Double-antibody Sandwich (DAS) ELISA are optimized to detect insecticidal protein in transgenic cry8Ca tobacco against Rutelidae larvae (Coleoptera:Scarabaeidae). As follows: the density of anti-rabbit immun serum and the enzyme(HRP)-labelled antibody were 1:3200 and 1:400 respectively, extracting buffer was pH 9.6 NaHCO3- Na2CO3, coating buffer was pH7.4 PBS, confining liquid was 0.5% Gelatin-PBST, TMB substrate action time was 15min. Cry8Ca protein in transgenic tobaccoes were detected by this DAS –ELISA. The roots of line S12 contain 12.683-14.461ug Cry8Ca protein per gram fresh weigh about 0.052%-0.062% of total protein more than others.

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