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

为了阐明某些疾病时急性期糖蛋白糖链结构的变化，本研究从单抗制备、L-α-选择素基因克隆及其表达、糖链分析、淋巴细胞功能分析等方面对牛α-1AGP进行了研究。单抗制备：从牛血清中提纯α1AGP，用于免疫小鼠。经免疫印迹分析，获得3株能识别牛α1AGP的单抗(含IgM轻链)。结论：1.Western印迹分析表明，经糖链切割后的单抗不与α1AGP反应。牛L-选择素基因克隆及其蛋白表达：克隆了牛L-选择素基因，其开放阅读框全长1110bp，编码370个氨基酸。在细菌表达中，重组蛋白为不溶蛋白。在杆状病毒-昆虫细胞的表达中，该蛋白以膜蛋白的形式表达，并与特异性抗体发生反应。糖链分析：正常人α1AGP可分离出两个与ConA亲和力较高的组分，而牛白血病患者除这两个组分外，还可分离出4个亲和力较低的组分。与E-SEL一样，抗唾液酸化Lewis X抗体对牛α1AGP的反应性较弱，但在牛白血病中的反应性似乎比正常健康人更强。牛淋巴细胞功能分析：对牛淋巴细胞进行细胞因子基因表达分析。IL-2mRNA在淋巴细胞中的表达存在个体差异，但IL-4表达降低，干扰素-γ表达升高。

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.