人共刺激分子4IgB7-H3与人β1，3半乳糖基转移酶7相互关系初探

发布时间：2018-01-13 17:06

本文关键词：人共刺激分子4IgB7-H3与人β1，3半乳糖基转移酶7相互关系初探　出处：《苏州大学》2008年硕士论文　论文类型：学位论文

【摘要】： 目的:β1,3半乳糖基转移酶7[β3GalT7(GnT8)]是我室通过电子克隆得到的一个新型的人类糖基转移酶。人共刺激分子4IgB7-H3为Ⅰ型跨膜糖蛋白,使用软件预测发现其氨基酸序列上有相当数量的糖基化位点。本文期望通过对4IgB7-H3与β3GalT7(GnT8)的相互关系进行研究,探索β3GalT7(GnT8)对B7-H3蛋白的翻译后修饰作用。方法:构建β3GalT7(GnT8)基因反义表达细胞,RT-PCR和Western blot研究其基因下调对人4IgB7-H3分子表达的影响;构建4IgB7-H3基因转染细胞,RT-PCR和Western blot研究其基因上调对人β3GalT7(GnT8)分子表达的影响;多种在线糖基转移酶作用位点预测软件联用,预测人4IgB7-H3氨基酸序列上潜在的糖基转移酶作用位点,免疫共沉淀研究两种蛋白质之间的相互作用,细胞免疫荧光化学术检测两种蛋白质在细胞内的共定位。结果:获得了β3GalT7(GnT8)基因反义表达稳定细胞株与高表达4IgB7-H3基因转染稳定细胞株;β3GalT7(GnT8)基因表达下调使4IgB7-H3基因表达受到一定程度的抑制;β3GalT7(GnT8)基因表达随着4IgB7-H3基因表达上调略有上升;软件预测4IgB7-H3氨基酸序列上潜在的O-GalNAc糖基化位点有3个,N-糖基化位点有8个,O-β-GlcNAc的附着位置有18个;β3GalT7(GnT8)蛋白与4IgB7-H3蛋白能共沉淀,并且两者能在细胞内共定位。结论:在mRNA和蛋白表达水平,β3GalT7(GnT8)和4IgB7-H3的表达呈正相关;β3GalT7(GnT8)蛋白和4IgB7-H3蛋白在氨基酸序列上和空间上具有相互作用的可能性;β3GalT7(GnT8)蛋白可能对4IgB7-H3蛋白有糖基化修饰作用。
[Abstract]:Objective: to investigate the effect of 尾 _ 1N _ 3-galactosyltransferase 7. [尾 _ 3GalT _ 7 GnT _ 8] is a novel human glycosyltransferase obtained by electronic cloning. The human costimulatory molecule 4IgB7-H3 is a type I transmembrane glycoprotein. A considerable number of glycosylation sites were found in the amino acid sequence by software prediction. The relationship between 4IgB7-H3 and 尾 3GalT7 GnT8) was expected to be studied. To explore the posttranslational modification of B7-H3 protein by 尾 _ 3GalT _ 7 GnT _ 8. Methods: 尾 _ 3GalT _ 7 GnT8) gene antisense expression cells were constructed. RT-PCR and Western blot were used to study the effect of down-regulation of its gene on the expression of human 4IgB7-H3 molecule. RT-PCR and Western blot were constructed to study the effect of up-regulation of 4IgB7-H3 gene on the expression of human 尾 _ 3GalT _ 7G _ nT _ 8. A variety of online glycosyltransferase interaction site prediction software was used to predict potential glycosyltransferase interaction sites in the amino acid sequence of human 4IgB7-H3. The interaction between the two proteins was studied by immunoprecipitation. Cellular immunofluorescence was used to detect the co-localization of two proteins in cells. Results: 尾 _ 3GalT _ 7G _ nT _ 8) gene antisense expression stable cell lines and high expression 4IgB7-H3 gene transfected stable cell lines were obtained. The down-regulation of 尾 _ 3GalT7-GnT8 gene resulted in the inhibition of 4IgB7-H3 gene expression to some extent. The expression of 尾 3GalT7 + GnT8) gene increased slightly with the up-regulation of 4IgB7-H3 gene. The potential O-GalNAc glycosylation sites in the 4IgB7-H3 amino acid sequence were predicted by software. There were 3 N-glycosylation sites and 8 O- 尾 -GlcNAc attachment sites in 4 IgB7-H3 amino acid sequences. 尾 _ 3GalT _ 7 GnT _ 8) protein and 4IgB _ 7-H _ 3 protein can co-precipitate and co-locate in cells. Conclusion: there was a positive correlation between the expression of 尾 3GalT7 (GnT8) and 4IgB7-H3 in the expression of mRNA and protein. 尾 _ 3GalT _ 7 GnT _ 8) protein and 4IgB _ 7-H _ 3 protein have the possibility of interacting with each other in amino acid sequence and space. 尾 _ 3GalT _ 7 GnT _ 8) may have glycosylation effect on 4IgB _ 7-H _ 3 protein.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R392

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