【作者】 严吉明；

【导师】 叶华智；

【作者基本信息】 四川农业大学 ， 植物病理学， 2002， 硕士

【摘要】 培育和利用抗虫品种是防治作物害虫最经济有效的措施。通过转抗虫基因培育抗虫品种是重要方法之一。此项工作中需要对转基因植物进行鉴定和筛选，因此发展快速、准确、简便的检测技术具有重要的作用和意义。本研究以检测目前抗虫转基因育种中研究最多、获得成效最大的转Bt抗虫基因的表达产物（杀虫蛋白）为检测目标。为此研究了影响抗原和抗体制备的主要因素，制备出灵敏度高的特异抗体，在此基础上开展检测方法研究，掌握影响检测效果的有关因素，从而初步建立起灵敏度高的ELISA检测技术。本研究取得如下结果： 抗原是影响抗体特异性和效价的重要因素，为了得到产量高、质量好的杀虫晶体蛋白作为抗原，我们研究了Bt菌培养时间、伴孢晶体蛋白裂解液的种类和处理温度与时间对杀虫蛋白提取的影响。结果表明Bt HD-1菌株在趴生长繁殖培养基（Singer等，1968）上，30℃下培养41-55h均可获得大量孢晶混合物。采用50mMNa₂CO₃（含50mM EDTA，3％巯基乙醇，pH10）裂解液和0.04N Na0H的裂解液处理孢晶混合物，均可获得大量的130KD杀虫蛋白。但用0.04N NaOH提取的杀虫蛋白中含有较多杂蛋白。所研究的处理温度和时间对杀虫蛋白提取量和组成无明显影响。 抗原的免疫剂量是影响抗体产生的一个重要因素，免疫剂量过大会引起麻痹反应。为了提高免疫效果，获得较好的抗体。我们采用两种抗原剂量（750ug／只.次和375 ug／只.次）免疫家兔，结果表明所供试的两种剂量对抗血清效价和特异性无明显影响。因此采用任一种剂量免疫家兔均可。本研究制备的抗体用于检测标准抗原，灵敏度可达7.8-16.5ng。 在建立检测方法研究上，采用ELISA间接检测法，测定了两种酶标羊抗兔抗体（辣根过氧化物酶与碱性磷酸酶）和两种载体（聚苯乙烯酶标板和硝酸纤维膜）对检测灵敏度和样本检测效果的影响。结果表明，在采用纯的杀虫蛋白进行灵敏度检测时，二种酶标抗体和两种检测载体的测定结果非常相近，无明显差别。但在对植物样本检测时，不同酶标抗体和载体则存在差异。表现为碱性磷酸酶标记的抗体优于辣根过氧化物酶标记的抗体。这主要是由于植物样本中存在有内源过氧化物酶的干扰。在两种检测载体上，由于植物样本中存在叶绿素，采用硝酸纤维膜进行Dot-ELISA检测时，叶绿素色斑干扰较大。如何排除叶绿素在D。t－ELISA检测中的影响，尚值得研究。 通过本研究，基本掌握了提取杀虫蛋白作为抗原和兔疫家兔制备抗血清的有关因素和条件，制备出了特异性强的抗体，检测灵敏度可达 7．8刁．6ng。通过检测方法研究，明确了影响检测的一些因素并初步建立起了检测棉叶样本中Bt杀虫蛋白的相关技术。更多还原

【Abstract】 Breeding and growing resistant culture was one of elements in the Integrated Pest Management (IPM). Genetic engineering can be used to breed resistant crop. The Bt gene producing insecticidal protein was transferred into cotton and other crops to improve crop resistance to pest.The goal of this research was to study on the preparation of Antigen of Bt insecticidal protein, production of antibody, and the test method technique for assay of the Bt toxin protein in transgenic cotton and other crop plants by using ELISA. The result was as follows: 1.Preparation of Bt insecticidalThe Bt isolate HD-1 was used in this study for producing toxin protein. The Bt bacterium was cultivated on solid medium at 30癈for 41-55hr. When the crystal protein formed and separated from bacterium cells, the colony on the plate was washed with water and centrifuged 5000g and 4 to collect the preparation of spore and crystal protein. The mixture of spore and crystal protein was washed three times with PBS pH7.4 and d-HaO and dissolved the crystal protein in alkaline solution(0.04N NaOH at 4癈 2-3hr or 50mM NaCO3,50mM EDTA,3% & -rnercaptoethanol,pH10 at 37癈 Ihr or 4癈 4hr) . The solution was centrifuged at SOOOg 20min. The supemate was collected and adjusted the pH to 4.4-5 with NaAc-HAc buffer pH4.4 or Acetic acid to precipitate the toxin protein overnight at 4癈. The precipitate of protein was gained by centrifuge at 5000g 20min 4癈 and washed three time. The protein was used for Antigen. 2.Antiserum of Bt productionAntibodies specific to Bt toxin protein were produced in six New Zealand white male rabbits by immunization procedure. The animals immunized with approximately 750ng or 375ng of antigen per/rabbit in Freund’s complete adjuvant. After 30 days of first immunization, subsequent immunizations were conducted using same dosage of Antigen in Freund’s incomplete adjuvant at 10 days intervals (three immunizations). Blood was drawn from the heart 10 days after last injection.Blood Samples were allowed to coagulate, then, after overnight storage in a refrigerator,-35-the serum was decanted and clarified by low speed centrifugation. The title of Antibody was tested by ouchterlony gel diffusion. In my experiment, the title was 1/80-1/120 depending on the individual of the rabbit. The IgG fraction from rabbit Antiserum was precipitate by salting out with ammonium sulphate. 3.Establishment of ELISA assay for detection of Bt toxin proteinTwo ELISA assay (indirect ELISA and Dot ELISA) were used in this research. The ELISA procedure consisted five general steps:1.Preparing the test samples and diluting standards antigen in 0.1% BSA, 0.05M PBST pH7.4, 1%PVP.2.Add test samples or antigen to plate well nitrocellulose membrane, incubate overnight at 4癈 or dry at room temperature for at least lh..3 .Block the plate well or membrane with PBS pH7.4 containing 10 mg/ml BSA.4.Add specific antibody to the plate well or membrane and incubate for 2-3h at room temperature.5.Add enzyme-labeled anti-rabbit Antibody and incubate for 2h at room temperature. Plate wells or membrane were washed between each step from 2-5. The washing was accomplished by 4-5 washes with PBST pH7.4, at room temperature.6.Add enzyme substrate to develop the color at room temperature and dark condition for about 30 min. Stop the reaction and evaluate the OD value.The detectable level of purified Bt toxin protein by indirect ELISA and Dot ELISA was 7.8-15.6ng. Five samples of Bt transgenic cotton and two contrastive cottons (non-transgenic cotton) were tested using indirect ELISA and Dot ELISA. The results showed that the two assays could be used for detecting Bt toxin protein in transgenic cotton, but some problems (such as interference of sample green color) need to be solved.更多还原