Preparation Of Monoclonal Antibodies From Mycelium Protein Of Setosphaeria Turcica

The HT-toxin from Setosphaeria turcica was found to be the most important toxigenic factor of Northern Leaf Blight of Corn in the former research in our laboratory. The possible pathogenic mechanism of S. turcica was also elucidated as the binding "receptors" of HT-toxin in the corn cell membrane, which could result in the change of membrane permeability and the activity of enzymes related to active oxygen (AO) and in the end induced the symptoms of the disease. However, because the HT-toxin was identified as a group of some low molecular weight compounds, it was very difficult to study the interaction between HT-toxin fractions and cell membrane proteins of corn with different Ht gene using immunochemical techniques. Utilizing the monoclonal antibody (Mb) of mycelium proteins connectively related to the production of HT-toxin or its specific fraction as research resources will provide a new way to study the host-pathogen interaction.A mycelium protein that was specific to the isolates of race 1 was isolated by the PAGE analysis of the mycelium proteins of race 0 and race 1 of S. turcica. The molecular weight of the protein was 25 KD with SDS-PAGE analysis. The 25 KD protein (the concentration of the protein is 173.8 ug/mL) of isolate 99-2 belong to race 1 was purified and chosen as the antigen to immune the 6-8 week old BALB/C mice. The antisera with the titer of 1:1600 were extracted and purified after 4 weeks. Then, the monoclonal antibodies were prepared by the cell fusion of mouse spleenocytes and myeloma cells, and at the same time 12 Mbs were identified by indirect ELISA.The cross reaction between the monoclonal antibodies and mycelium proteins of race o and race 1 were proceeded and the results had shown that one monoclonal antibody (Mb4) was the most specific to isolate 99-2, 7 monoclonal antibodies (Mb1, Mb2, Mb3, Mb5, Mb6, Mb10, Mb11) only reacted with mycelium proteins of race 1,and the other 4 monoclonal antibodies (Mb7, Mb8, Mb9 and Mb 12) could react to mycelium proteins of both race 0 and race 1. Moreover, 4 monoclonal antibodies (Mb2, Mb3, Mb8 and Mb9) could react to HT-toxin of isolate 99-2, which indicating that the monoclonal antibodies could be used to study the possible characteristics of HT-toxin and its fractions.The difference between race 0 and race 1 was not only shown in their pathogenicity, the composition of HT-toxin, but also in the constitution of mycelium proteins. The specific 25 KD mycelium protein isolated in this experiment held high possibility related to the production of HT-toxin from race 1 of S. turcica.