Primary Study Of Relativity Between Human Co-stimulative Molecule 4IgB7-H3 And Human UDP-Gal: BetaGlcNAc Beta 1, 3-galactosyltransferase Polypeptide 7

Object:UDP-Gal:betaGlcNAc beta 1,3-galactosyltransferase polypeptide 7 [β3GalT7(GnT8)] is a new member of glycosyltransferases we gained by electric cloning.The costimulatory molecule human 4IgB7-H3 is aⅠtype transmembrane glycoprotein.We found many glycosylation sites in 4IgB7-H3 sequence by using some softs.This thesis aims to study the relativity between 4IgB7-H3 andβ3GalT7(GnT8) and research the role ofβ3GalT7(GnT8) in the process of B7-H3 protein post-translational modification.Methods:The recombinant plasmid pEGFP-C1-T7-Antisence was transfected into SGC7901 and established by means of G418 selection.The recombinant vector pEGZ-Term-4IgB7-H3 and two helper virus vectors were cotransfected into the package cell with Lipofectamine 2000.The supernatant was used to infect L929 cells.RT-PCR and Western blot were used to study the expression of 4IgB7-H3 andβ3GalT7(GnT8) on mRNA and protein level.Several online softs were used to predicted glycosylation site in 4IgB7-H3 sequence.Co-immunoprecipitation was used to study the combination of 4IgB7-H3 protein andβ3GalT7(GnT8) protein.Immunofluorescence was used to locat the two proteins.Results:SGC7901/T7-AS cell line and SGC7901/4IgB7-H3 cell line were successfully obtained.The expression of 4IgB7-H3 was inhibited follow the down-regulation ofβ3GalT7(GnT8).The expression ofβ3GalT7(GnT8) was up-regulated follow the up-regulation of 4IgB7-H3 on mRNA and protein level. There are 3 O-glycosylation sites,8 N-glycosylation sites and 18 O-(beta)-GlcNAc sites in 4IgB7-H3 sequence.4IgB7-H3 protein andβ3GalT7(GnT8) protein can combine together and mutual locate in cell. Conclusion:The expression of 4IgB7-H3 andβ3GalT7(GnT8) was positive correlation. We found 4IgB7-H3 protein andβ3GalT7(GnT8) may interact by analyzing 4IgB7-H3 sequence and their location in A549 cell.β3GalT7(GnT8) may modify 4IgB7-H3 protein.