ANALYSIS OF GLYCAN CHAIN AND BINDING AFFINITY TO ADHESION MOLECULES ON ACUTE PHASE GLYCOPROTEIN IN BOVINE LEUKEMIA

牛白血病急性期糖蛋白聚糖链及粘附分子结合力分析

• 批准号: 12660285
• 负责人: IWATA Hiroyuki
• 金额: $ 0.7万
• 依托单位: YAMAGUCHI UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2000
• 资助国家: 日本
• 起止时间: 2000 至 2001
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-12660285/
• 关键词: α1-acid glycoprotein; monoclonal antibody; L-selectin; glycan chain; bovine leukemia; E-selecin; sialyl lewis X antigen; シアソルルイスX抗原

In order to clarify the changes of glycan chain structure of acute phase glycoproteins in certain diseases, bovine α 1-acid glycoprotein ( α1AGP ) was studied on the preparation of monoclonal antybody ( Mab ), cDNA cloning of L-selectin and its expression, analysis of glycan chain, and functional analysis of lymphocytes.1. Preparation of monoclonal antibody: α1AGP was purified from bovine sera and used for immunogen to mice. Three Mabs obtained ( IgM with κ light chain ) recognized bovine α1AGP on Western blot analysis. The Mabs was not reacted with α1AGP after cleavage with glycan chain on Western blot analysis.2. cDNA cloning of bovine L-selectin and its protein expression: Bovine L-sel cDNA was cloned and its ORF was 1110 bp which encode 370 amino acids. In bacterial expression, the recombinat protein was insoluble. In baculovirus-insect cell expression the protein was expressed as membrane proteins, which reacted with the specific antibody.3. Analysis of glycan chain: α1AGP was resolved into two fractions with high affinity to ConA in normal health, but 4 fractions with lower affinity in addition to the two fractions in bovine leukemia. As well as E-sel, the reactivity of antibody against sialyl Lewis X to bovine α1AGP was weak, but seemed to be stronger in bovine leukemia than in normal health.4. Functional analysis of bovine lymphocytes: Cytokine mRNA was analyzed on bovine lymphocytes. IL-2 mRNA expression in lymphocytes vary in individuals, but IL-4 was decreased, and IFN-γ was increased with individual differences.

为了阐明急性期糖蛋白糖链结构在某些疾病中的变化，本研究对牛α 1酸性糖蛋白（α1AGP）进行了单克隆抗体（Mab）的制备、L-选择素cDNA的克隆及表达、糖链分析和淋巴细胞功能分析.单克隆抗体的制备：从牛血清中纯化α1AGP，作为免疫原免疫小鼠。在Western blot分析中，获得的三种单克隆抗体（IgM和κ轻链）识别牛α 1 AGP。Western blot分析表明，单克隆抗体经糖链切割后，不与α1AGP发生反应.牛L-选择素的cDNA克隆及蛋白表达：克隆了牛L-选择素的cDNA，其开放阅读框为1110 bp，编码370个氨基酸。在细菌表达中，重组蛋白是不溶性的。在杆状病毒-昆虫细胞表达中，该蛋白以膜蛋白的形式表达，并与特异性抗体发生反应.聚糖链分析：正常牛血清中α1AGP被分离成两个与ConA亲和力高的组分，而牛白血病血清中除这两个组分外，还分离出4个与ConA亲和力低的组分。与E-sel一样，抗唾液酸刘易斯X抗体与牛α 1 AGP的反应性也很弱，但在牛白血病中的反应性似乎比正常人强.牛淋巴细胞的功能分析：在牛淋巴细胞上分析细胞因子mRNA。淋巴细胞IL-2 mRNA表达量在个体间存在差异，但IL-4表达量降低，IFN-γ表达量升高，且存在个体差异。

项目成果

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Eckersall, P.D., 岩田祐之他: "急性相反応"Jpn J. Clin. Path.. 5. 15-22 (1999)

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Iwata,H. et al.: "Monoclonal Antibody against Bovine α_1-Acid Glycoprotein"Journal of Veterinary Medical Science. 62・10. 1099-1100 (2000)

Iwata，H.等：“抗牛α_1-酸性糖蛋白的单克隆抗体”兽医医学杂志62·10（2000）。