| In this research,purified soybean agglutinin (SBA) were used to study the stability to pepsin and trypsin degradation in vitro,which weretwo principal proteinases in animal bodies.Proteolytic degree of SBA were analyzed by method of sodium dodecyl sulfate polyacrylamide gel electropheresis(SDS-PAGE). Variation of immunocompetence of SBA were detected by indirect competitive Enzyme-Linked Immunosorbent Assay (ELISA) in process of proteolysis.Purified SBA was isolated by a N-acetyl-galacotsamine-epoxy-actived sepharose 6B Affinity charomatography system. Female white rabbits weighing about 3 kg were used for obtaining polyclonal antibodies to the immunogens (SBA). Serum was isolated through centrifugation and purified by Saturated Ammonium Sulfate method.An indirect competitive ELISA methed of SBA was developed with polyclonal antibodies and purified SBA. This assay was applied to analyze immunocompetence of SBA in process of proteolysis.In the experiment, SBA was hydrolyzed by pepsin and trypsin at five concentration gradients(1mg/mL,2mg/mL,3mg/mL,4mg/mL and 5mg/mL) and nine time gradients (15s,30s,60s,2min,4min, Smin, 15min,30min and 60min ).The results were performed by General Linear Model of SPSSI0.0 software package.In addition,the activity of protease must keep stable during the experiment,so the activity was often monitored according to mensuration of enzyme activity in China pharmacopeia of 2005.The results of SBA degraded by pepsin showed that SBA can be hydrolyzed by pepsin slowly. The time of complete degradation was prolonged with concentration of SBA becoming higher. Furthermore, during the process of proteolysis,immunocompetence of SBA was becoming little and vanished at last. the speed of disappearance of immunocompetence became slow with the time of enzymolysis extended.the higher concentration of SBA was,the faster immunocompetence of SBA diminished.but,the time of complete disappearance of immunocompetence was prolonged.The results of SBA degraded by trypsin showed that SBA can not be hydrolyzed by trypsin. Furthermore,immunocompetence of SBA was unaffected in the process of enzymolysis.These demonstrated that SBA had high stability to enzymolysis of trypsin. Based upon research above, we obtained following conclusions. SBA can be degraded by pepsin,but the process of enzymolysis needed comparatively long time and necessary pepsin concentration. If SBA was not completely degradated in stomach, the remaining can not be degradated by trypsin in intestinal tract.The remaining could be harmful to animals as antinutritional factors, which had negative effects to digestion and utilization of nutrients. Because SBA can bind to the specificity receptors of small intestinal mucosa epithelium and enter epithelium by internalization ,thus effect digestion and absorptive function of small intestinal. Some SBA got into circulation system through small intestinal mucosa epithelium and was harmful to animal organism.Therefore, it is necessary to inactivate the activity of SBA in food and feedstuff and detect remaining of SBA strictly by national quanlity organization. |
