Purification And Characterization Of Two Inhibitors Of Helicoverpa Cathepsin B-Like Proteinase

There are many proteinase inhibitors (PIs) in tissues of animals and plants, and they have inhibitory activity to proteinase. As we all know, Helicoverpa armigera cathepsin B (HCB) inhibitors are very important PIs. Many studies show that the plants containing cathepsin B inhibitors, can inhibit the activity of cathepsin B in insects, thus the embryo can not get enough nutrition, and it will die. Therefore, the studies of HCB inhibitors have benign using prospect.As an important pest in agriculture, Helicoverpa armigera does great harm to the production of cotton. HCB is exprssed in imago of Helicoverpa armigera, and it is involved in the degradation of fat badies to provide nutrition for the embryonic development, so HCB can become a new target for controlling insects. Nowdays, the researches, no matter in country or aboard, are mainly about the inhibitors of serine proteinase. But there are no reports about the application of cysteine proteinase in developing embryo for using gene-transform plants, and the discovery of HCB-SunI and HCB-SoyI may supply a new method for the research of gene-transform plants.The studies of our work show that many plants, such as sunflower seed, soybean, cotton seed and rice, have HCB inhibitors with great inhibitory activity. On the base of these researchs, using the conventional biochemical analysis methods and chromatography, two inhibitors against HCB named HCB-SunI and HCB-SoyI were purified from sunflower seeds and soybean.First, we used 50% (NH₄)₂SO₄ to precipitate protain, then we got the pure inhibitors after Sephadex G-75 and DEAE-Toyopearl chromatography. The results of 15% SDS-PAGE electrophoresis showed that they are low molecular weight (LMW) proteins. The studies of biochemical characters of HCB-SunI and HCB-SoyI show that they can inhibit the activity of HCB to BSA in acid conditions; they can inhibit the growth and development of Geliomas and HeLa cells. Besides, HCB-SunI has great stability to temperature and pH.Besides, HCB inhibitors can inhibit the growth, transfer and soakage of the tumors. Because the inhibitors of HCB are generally efficient at reducing invasion, HCB is thought to play an important role in murine melanoma cell invasion. Thus, Gene-transform plants can produce Pis to lessen the endanger of the pest. The results of this paper promised that the inhibitor would have a prospect of developing a transgenetic insect resistance plant or developing a tumor therapy.