Isolation And Identification Of Soybean Agglutinin Specific Binding Proteins On Erythrocyte Membranes Of Different Species Of Animals

Soybean agglutinin(SBA)is a kind of protein with high biological activity in soybean.SBA exerts its functions mainly through the direct or indirect connection with cells,and one of its most basically biological functions is the ability to agglutinate erythrocyte.It has been shown that the sensitivity of the erythrocyte to SBA is different in different animals species.In this study,the erythrocyte of dairy cows,pigs and rabbits were selected as the experimental materials to study the differences of SBA binding proteins on the erythrocyte membrane of different animals.Experiment 1: The erythrocyte membranes of bovine were extracted by the method of hypotonic hemolysis with NP-40 lysis solution,RIPA lysis solution(medium)and RIPA lysis solution(weak),respectively.The results showed that the effect of NP-40 lysis on the erythrocyte membrane protein was significantly better than that of RIPA lysis solution(medium)and RIPA lysis solution(weak).The SDS-PAGE results of erythrocyte membrane proteins in dairy cows,pigs and rabbits revealed that the composition of the erythrocyte membrane proteins in these three animals were similar.Most of the proteins were distributed at 25 kDa-250 kDa,and the proportion of proteins with high molecular weight were significantly higher than that of low molecular weight proteins.However,there also had some differences: the erythrocyte membrane protein was the most abundant in cow.The protein contents of rabbit erythrocyte membrane proteins in 180 kDa and 45 kDa were lower than that of cows and pigs,but the protein abundance of erythrocyte membranes in porcine at 35 kDa was lower than that of cow and rabbit.Experiment 2: the target proteins were obtained by Co-Immunoprecipitation(Co-IP)method,and then the target proteins were identified by liquid chromatography mass spectrometry / mass spectrometry(LC-MS/MS).The results were retrievaled using the MASCOT(version number: 2.3.0)software.The results of MASCOT showed that the protein score was higher than 24 in cow,higher than 23 in pig,and higher than 22 in rabbit were considered as the positive results.The higher the score,the higher the credibility.According to the functions of the identified proteins,the erythrocyte membrane proteins of each animal can be divided into three types: cytoskeletal proteins,catalytic enzyme proteins,and functional regulatory proteins.The skeleton proteins and their score were higher than catalytic enzyme proteins,and functional regulatory proteins in the same species.The same proteins were also observed in different animals,but most proteins have species differences.The results provide an important experimental evidence for studying the difference of sensitivity to SBA in different animals species...