| 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.
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