| 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...
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