研究肠道糖基化修饰的荧光凝集素免疫组化方法建立

doi:10.16431/j.cnki.1671-7236.2020.07.007

Abstract

Abstract: Glycosylation is one of the most important post-translational modifications of proteins in life and plays a very important role in life.Oligosaccharides on glycoproteins can serve as binding sites for lectins,so lectins can be used to identify and analyze sugar chain structures,as an important complement to glycosylation analysis methods such as mass spectrometry.The purpose of this study was to establish and optimize the fluorescence lectin-based immunohistochemical method to study the glycosylation modification in the pig intestines,and to apply this method to study the differences in glycosylation modification in different intestinal segments and the effect of weaning on intestinal glycosylation modification in piglets.The results showed that:①The optimal conditions for fluorescence lectin-based immunohistochemical method were:deparaffinized sections were blocked with carbohydrate blocking solution for 30 min,sections were then incubated with 5 μg/mL FITC-lectin for 1 h at room temperature,and mounted with DAPI mounting medium.This method was characterized with high sensitivity,good specificity and more diversification regarding quantification.②Different glycosylation modification patterns were observed in the ileum and colon of growing pigs.The ileum was enriched with fucose and N-acetylglucosamine,while the colon was enriched with N-acetylglucosamine,fucose and mannose.Except that mannose was distributed in the non-goblet cells in the intestinal epithelium and lamina propria,fucose and N-acetylglucosamine were mainly distributed in the surface of intestinal villi and goblet cells.③The content of N-acetylglucosamine in the ileum increased along with the age of 22-28 days old piglets (P<0.05),and the content of N-acetylglucosamine in the ileum of piglets was significantly reduced by weaning at day 28 (P<0.05).In conclusion,a method to investigate glycosylation modification in pig intestine was successfully established in this study,and it was found that N-acetylglucosamine in the ileum could be used as a biomarker to indict a health intestinal barrier function in piglets by this method.

Key words: pig; intestine; glycosylation; lectin; fluorescent immunochemistry

CLC Number:

Q493.99

WEN Xiaobin, XIA Bing, XIE Jingjing, FENG Xiaohui, WU Weida, LIU Lei, ZHANG Hongfu. Establishment of Fluorescent Lectin-based Immunohistochemical Method to Investigate Glycosylation in the Intestine[J]. China Animal Husbandry and Veterinary Medicine, 2020, 47(7): 2025-2032.

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Establishment of Fluorescent Lectin-based Immunohistochemical Method to Investigate Glycosylation in the Intestine

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