Interacting Protein In Human Tumor Cell Of Bovine Beta-Lactoglobulin

Beta-lactoglobulin (β-lactoglobulin, β-LG) is a major whey protein of bovine milk, which belongs to the lipocalin family. β-lactoglobulin can work as a carrier for many small hydrophobic substances such as retinol,β-carotene, Vitamin D, therefore, it will be a potential tool for oral colon-specific drug delivery system (OCDDS). However, the researches about p-LG receptors were rarely, some were on NT2 cells, MARK-3 cells Erythrocyte, Germ cells. Further more, the research about β-lactoglobulin receptor on intestinal cells has not been found yet. For this reason, this paper carried out the research of β-lactoglobulin receptor on intestinal cells with a strategy involving co-immunoprecipitation (Co-IP), one-dimensional sodium dodecyl sulfonate-polyacrylamide gelelectrophoresis (1DE-SDS-PAGE), and capillary nano liquid chromatography/tandem mass spectrometry (nanoLC-MS/MS).Caco-2 cells and LoVo cells both were cultured and used for our study, the results showed that Caco-2 cells patchy distribution and need 72 h to cover the whole bottom of cell culture flask. LoVo cells have a regular shape and need 48 h to cover the entire bottom. fluorescence probes were then used to mark the cell membrane, nuclar and P-LG, respectively. Confocal laser scanning microscopy was used to detect the interactin between β-LG and the cells at Omin、15min、30min、60min. The results showed that, after being incubated with Caco-2 cells and LoVo cells, respectively, for 1h period. β-LG was enrichment at the outside of the membrane, but didn’t inter the cells.After being incubated for 1h, β-LG and its interacting proteins were separated by co-immunoprecipitation (Co-IP), SDSPAGE, Coomassie blue staining and Western Blotting were used to detect the β-LG and its interacting proteins captured by Co-IP technology. The results showed that two brands between 20kDa and 70 kDa could be seen. Coomassie blue staining could not detect β-LG in the gel. Western Blotting detected that β-LG existed in the Co-IP sample.Silver staining and nanoLiquid Chromatography-Mass/Mass (nanoLC-MS/MS) were used to analyze P-LG and its interacting proteins from Caco-2 cells or LoVo cells. The results showed that besides β-LG, other 3 proteins (40S ribosomal protein S25,40S ribosomal protein S18, histone H4) were also identified; however, there was not any cell membrane receptors can be detected. Those 3 proteins maybe interact with β-lactoglobulin inside the cells.