【作者】 王清；

【导师】 吴振廷；

【作者基本信息】 安徽农业大学 ， 农业昆虫与害虫防治， 2005， 硕士

【摘要】 转 Bt 基因棉抗虫性的检测是转 Bt 基因棉的研究、选育和种子生产的重要环节,对保证种质纯度有重要意义,建立检测灵敏度高、操作简便的快速检测方法是当前急待解决的问题。本次研究的主要目的是建立快速检测 Bt 毒蛋白的胶体金免疫检测技术。其结果如下: 1.Bt 毒蛋白的分离与纯化及其多克隆抗体的制备 本研究从苏云金芽孢杆菌库斯塔克亚种 HD-73 中分离提取 Bt 前毒素蛋白,利用碱裂解法,酸沉淀粗提前毒素蛋白,再经胰蛋白酶水解,凝胶层析柱纯化得到电泳纯Bt 毒蛋白,并以此为抗原免疫兔子,从兔抗 Bt 毒蛋白血清中分离纯化多克隆抗体,为进一步的 Bt 毒蛋白的免疫检测奠定基础。 2.胶体金及金标抗体的制备 胶体金制备方法较多,可以根据不同粒径需要选择适当的方法。本次试验采用柠檬酸三钠还原法制备 20-30nm 的胶体金,用可见光光谱法对本次试验制备的胶体金进行鉴定,结果显示紫外分光光度计 450-800nm 扫描曲线光滑,峰宽较窄,证明胶体金质量合格。稳定 1ml 胶体金的最低蛋白质的量为 20μg 兔抗 Bt 毒蛋白多克隆抗体。制备的金标抗体经质量和稳定性鉴定均合格。 3.检测 Bt 毒蛋白免疫层析试验模型的建立 本研究建立了检测 Bt 毒蛋白免疫层析试验的模型,其检测 Bt 毒蛋白免疫层析条的制作方法:玻璃纤维素于 5ml 金标 Bt 毒蛋白多克隆抗体中浸泡 10 分钟后 37℃烤干备用,在Millpore层析速率为135sec/4cm硝酸纤维素膜上包被检测线和质控线,其中检测线为兔抗 Bt 毒蛋白多克隆抗体,质控线为羊抗兔抗体。然后组装试纸条:将吸水垫,硝酸纤维素膜,玻璃纤维素条依次粘好,相连部分相互重叠 2mm,整个试纸条的前后各粘一层黄色胶带,将前、后端上下包封住,用切割器切成 4mm 宽的条带。密封置干燥器内 4℃保存备用。样品处理的缓冲液为 0.05mol, pH9.5 碳酸缓冲液及使用体积为 150μl。 试纸条具体操作步骤如下:更多还原

【Abstract】 Testing the identification of resistance to cotton boll worm of Bt-transgenic cotton is important to the research of Bt-transgenic cotton, select the good Bt-transgenic cotton and production of Bt-transgenic cotton seeds. It is high time to establish a sensitive and simple method to test the identification of resistance to cotton boll worm of Bt-transgenic cotton. The aim of this study was to establish the immunogold biochemical and immune determination. The main results are summarized. 1. Purification of insecticidal toxin and preparation of its polyclonal antibody A polypeptide with a molecular weight of 66.2 kDa estimated by SDS-PAGE was purified as follows: the mixture of spores and crystals from Bacillus thuringiensis strain HD-73 was first solubilized with alkaline buffer and precipitated again with acid, then followed by trypsinisation and gel filtration chromatography. The purified polypeptide showing toxic activity to Helicoverpa armigera by ingestion was proved to be Bt toxin. Antiserum was obtained from rabbits immunized with this polypeptide as antigen. After further purification, polyclonal antibody of Bt toxin was got. Thus, the basis for determination of Bt toxin would be founded. 2. Preparation of colloidal gold and immunogold Although there were so many preparation methods of colloidal gold, proper method could be chosen according to granule diameter. In this trial, citromalic acid trisodium recovery method was employed to prepare 20-30nm colloidal gold. Ultraviolet spectrophotometer was used to determine the quality of colloidal gold. Scan outcome of colloidal gold between 450 and 800nm with ultraviolet spectrophotometer was used to appraise the quality of colloidal gold, the curve was clean and the width of hump was narrow. The result showed that the quality of colloidal gold was good. Trials of least protein protection amount was 20μg/ml . It was found that the stability and quality immunogold was satisfied though detecting. 3. Immunochromatographic assay detecting the Bt toxin In this trial, the model of detecting Bt toxin by immunochromatographic assay was established. The immunochromatographic test strips were made as follows: class fibers were dipped in 5ml immunoglod for 10min, then baked at 37℃. Nitrocellulose (NC) membrane with a flow time of 135 seconds/4 cm coated with polyclonal antibody of Bt toxin (as test line) and polyclonal antibody of goat anti rabbit (as control line). The process of assembling the test strips involved that absorb pads, NC membranes, class fibers were adhibited in turn and the adjoining parts were overlapped 2mm. Last, two ends of the test strips were covered with yellow adhesive tape, then cut into 4mm strips. It was necessary that the completed test strips were dryly conserved at 4℃. The sample was dilated with 50 mmo l·L - 1 carbonate buffer (pH9. 5) and volume of the buffer was 150μl. The test strips can be used as the following steps: first, the test strips should be taken out from 4℃ circumstance to room temperature; second, the end of the test strip which is conjugate pad was inserted into the sample; third, we can read the result after 10min to 20min. Positive results were developed with two visible red lines, while negative results only one visible red line which is control line. If there were no red lines, the test strip was unefficient. The whole process was completed with in less than 20 minutes without extra appliance and device. Further study was carried out to test the sensitivity of the immunochromatographic test strips. The results showed that the minimal detectable concentration of purified Bt toxin was 10ng/ml. The test strips can be dryly preserved at 4℃ for 3 months effectively. 4. Dot immunogold assay detecting the Bt toxin In this trial, the model of detecting Bt toxin by dot immunogold assay was established. The particular process was that firstly, the grids of 0.8cm×0.8cm were drawn on the Nitrocellulose (NC) membrane from Millipore Company; secondly, 1μl sample was coated in the center of one grid, and dried at更多还原