本文關(guān)鍵詞：人共刺激分子4IgB7-H3與人β1，3半乳糖基轉(zhuǎn)移酶7相互關(guān)系初探　出處：《蘇州大學》2008年碩士論文　論文類型：學位論文

  更多相關(guān)文章： β3GalT7 β3GnT 4IgB7-H3 糖基化位點預(yù)測 免疫共沉淀 細胞共定位

【摘要】： 目的:β1,3半乳糖基轉(zhuǎn)移酶7[β3GalT7(GnT8)]是我室通過電子克隆得到的一個新型的人類糖基轉(zhuǎn)移酶。人共刺激分子4IgB7-H3為Ⅰ型跨膜糖蛋白,使用軟件預(yù)測發(fā)現(xiàn)其氨基酸序列上有相當數(shù)量的糖基化位點。本文期望通過對4IgB7-H3與β3GalT7(GnT8)的相互關(guān)系進行研究,探索β3GalT7(GnT8)對B7-H3蛋白的翻譯后修飾作用。 方法:構(gòu)建β3GalT7(GnT8)基因反義表達細胞,RT-PCR和Western blot研究其基因下調(diào)對人4IgB7-H3分子表達的影響;構(gòu)建4IgB7-H3基因轉(zhuǎn)染細胞,RT-PCR和Western blot研究其基因上調(diào)對人β3GalT7(GnT8)分子表達的影響;多種在線糖基轉(zhuǎn)移酶作用位點預(yù)測軟件聯(lián)用,預(yù)測人4IgB7-H3氨基酸序列上潛在的糖基轉(zhuǎn)移酶作用位點,免疫共沉淀研究兩種蛋白質(zhì)之間的相互作用,細胞免疫熒光化學術(shù)檢測兩種蛋白質(zhì)在細胞內(nèi)的共定位。 結(jié)果:獲得了β3GalT7(GnT8)基因反義表達穩(wěn)定細胞株與高表達4IgB7-H3基因轉(zhuǎn)染穩(wěn)定細胞株;β3GalT7(GnT8)基因表達下調(diào)使4IgB7-H3基因表達受到一定程度的抑制;β3GalT7(GnT8)基因表達隨著4IgB7-H3基因表達上調(diào)略有上升;軟件預(yù)測4IgB7-H3氨基酸序列上潛在的O-GalNAc糖基化位點有3個,N-糖基化位點有8個,O-β-GlcNAc的附著位置有18個;β3GalT7(GnT8)蛋白與4IgB7-H3蛋白能共沉淀,并且兩者能在細胞內(nèi)共定位。 結(jié)論:在mRNA和蛋白表達水平,β3GalT7(GnT8)和4IgB7-H3的表達呈正相關(guān);β3GalT7(GnT8)蛋白和4IgB7-H3蛋白在氨基酸序列上和空間上具有相互作用的可能性;β3GalT7(GnT8)蛋白可能對4IgB7-H3蛋白有糖基化修飾作用。
[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.
【學位授予單位】：蘇州大學
【學位級別】：碩士
【學位授予年份】：2008
【分類號】：R392

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