| 苏云金芽胞杆菌杀虫晶体蛋白CryIA单克隆抗体的制备及在转Bt基因棉毒蛋白检测上的应用 |
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| 引用本文： | 王保民,何钟佩,田晓莉.苏云金芽胞杆菌杀虫晶体蛋白CryIA单克隆抗体的制备及在转Bt基因棉毒蛋白检测上的应用[J].棉花学报,2000,21(1):34-39. |
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| 作者姓名： | 王保民 何钟佩 田晓莉 |
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| 作者单位： | 中国农业大学作物化控中心,北京,100094 |
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| 摘 要： | 用苏云金杆菌菌株HD-1和HD-73分别发酵培养制备了孢晶混合物，孢晶混合物经分离、纯化、电泳得到CryIA、CryIA(c)杀虫晶体蛋白。用CryIA和CryIA(c)分别免疫家兔制备了两种多抗。用CryIA免疫小鼠，经细胞杂交、融合、克隆后筛选出4个单克隆株系A4F5F11、B4E6C7、B4E6D8、B4F5G11。用CryIA(c)多抗包被，B4E6D8上清液作为夹心抗体成功地建立了双抗夹心 ELISA，并检测了中棉所30、NUCOTN33B蕾期叶片中毒蛋白的含量，结果分别为23．4ng·g-1 FW、24．7ng·g-1 FW。
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| 关 键 词： | 转Bt基因棉 杀虫晶体蛋白 单克隆抗体 含量 |
| 文章编号： | 1002-7807(2000)01-0034-05 |
| 修稿时间： | 1999-05-20 |
Preparation of Monoclonal Antibodies of CryIA Insecticidal Crystal Protein and Its Application in Determining Toxin Level in Bt Cotton |
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| WANG Bao-min,HE Zhong-pei,TIAN Xiao-li.Preparation of Monoclonal Antibodies of CryIA Insecticidal Crystal Protein and Its Application in Determining Toxin Level in Bt Cotton[J].Cotton Science,2000,21(1):34-39. |
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| Authors: | WANG Bao-min HE Zhong-pei TIAN Xiao-li |
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| Institution: | WANG Bao-min
,HE Zhong-pei
,TIAN Xiao-li
;(Center of Crop Chemical Control of China Agricultural University, Beijing 100094,China) |
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| Abstract: | The spore/crystal complex was pre pared from Bacillus thuringiensis var. kurstaki HD-1 and HD-73 during sporulation. The parasporal crystal was isolated from the spore crystal/complex by liquid two phased methods. CryIA and CryIA (c) were precipitated from parasporal crystal on PI4.4. The 130～140 kDa molecular weight protein was separated with sodium-dodecy-sulfate polyacrylamide gel (SDS - PAGE ) electrophoresis. Two polyclonal antibodies were acquired using CrylA , CryIA (c)as antigens. ELISA titer of the two antisera were＞ 1/100000, ＞ 1/100000, respectively. Four monoclonal antibodies ( A4FSF11, B4E6C7, B4E6D8 and B4FSG11) were produced using CryIA insecticidal crystal protein (ICP) to immune BALB/C mouse. A double sandwich ELISA coated with anti-CryIA (c) polyclonal antibody was established with B4E6D8 as sandwich antibody. The toxin level of NUCOTN33B and CCRI-30 in leaf was detected. The content was 23. 4 and 24. 7 ng·g-1 FW,respectively. The result was identical with the insecticidal effect. |
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| Keywords: | Bt cotton ICP monoclonal antibody level |
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