Comparison Of Different Methods To Get Protein And Structure Analysis Of N-Linked Glycosylated Oligosaccharides Of Porcine Milk

In the production of modern swine industry,in order to improve sow production performance,pig slaughter rate and litter utilization,we usually need early weaning piglets.However,premature weaning of piglets is likely to cause piglet intestinal health damage,digestive system dysfunction,diarrhea,and even cause serious piglet death.How to reduce intestinal damage caused by early weaning has been the focus of research at home and abroad.And studies have found that postprandial weaning delay can avoid the early weaning stress caused by intestinal columnar cell activation induced mucosal injury.In the previous reports on human milk and bovine milk,we found that oligosaccharides have an important role in regulating intestinal flora and maintaining intestinal health,and that pig lactose may also be active in addressing intestinal damage caused by early weaning of piglets effect.The functions of oligosaccharides depend on their structures.To analyze the functions of oligosaccharides,it is necessary to analyze their structures accurately.However,almost all the research on oligosaccharides so far focused on human milk and bovine milk,and only the reports about porcine oligosaccharides are almost always associated with free oligosaccharides,and there is little research on glycosylated oligosaccharides.The glycosylated oligosaccharides,molecules have a variety of biological functions,and many of the glycoprotein function has also been confirmed to be linked to the oligosaccharides,so in-depth study of pig lactose will solve the weaned piglets health problems.In this study,the peak components of N-linked oligosaccharides in porcine milk were analyzed by high performance liquid chromatography(HPLC)combined with MALDI-TOF,compared the amount of two different kinds of porcine milk,and the amount of N-linked oligosaccharides of different lactation,then discussed the structure of N-linked oligosaccharides of each peak.1.Optimization of the method of obtaining N-linked oligosaccharideThe method of analyzing glycosylated oligosaccharides' structure involves a lot of steps and technique,the first part is mainly about optimization of glycoprotein separation.Several methods were used:acid precipitation method,organic reagent precipitation method,membrane separation method and ammonium sulfate precipitation method.Result showed that using 40%trichloroacetic acid(TCA)can get the maximum amount of glycoprotein,and then digested them by endonuclease PNGase F,then detected N-linked oligosaccharides by the high performance liquid chromatography(HPLC),we found that 40%TCA can also get the most N-linked oligosaccharides Therefore,in the next experiment,we using 40%trichloroacetic acid(TCA)precipitation method to obtain glycoprotein.2.Analysis of the structure of N-linked oligosaccharides from porcine milk.1)Analysis of N-linked-oligosaccharide Structure in porcine milk.In this chapter,the structure of N-linked oligosaccharide was analyzed by the oligosaccharide structure analysis platform established in the laboratory before.Firstly we used PNGase F to digest N-linked oligosaccharides,and then labeled it by 2-AB,then detected it by HPLC,the elution time of the HPLC peak component was converted into the standard GU value.Because different Monosaccharide has different GU value,we can reversing the structure of the possible oligosaccharides corresponding to each peak.And then detected each peaks by MALDI-TOF,the results showed that there were 60 possible structures in the N-linked oligosaccharides of porcine milk,and 17 structures were detected by MALDI-TOF-MS.12 of them were verified by MALDI-TOF-MS/MS.2)Compare the amount of N-linked oligosaccharides of different kinds of porcine milk in the same lactation,and compare the amount of N-linked oligosaccharides of the same porcine milk in different lactation.In this paper,the porcine milk we used were two different kinds of pigs'(Da bai and Mei shan)after piglets were produced 1 day/3 days/14 days and 21 days,The results showed that there were no differences in the structure of the N-linked-oligosaccharides between the two cultivars,but the amount were different.The total amount of N-linked oligosaccharides in the white pig was more than that of Meishan pig.With the prolongation of lactation,the total amount of N-linked oligosaccharide group of two breeds was declining.