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

为了阐明某些疾病急性期糖蛋白聚糖链结构的变化，对牛α1 -酸性糖蛋白（α 1agp）进行了单克隆抗体（Mab）制备、l -选择素cDNA克隆及其表达、聚糖链分析和淋巴细胞功能分析等方面的研究。单克隆抗体的制备：从牛血清中纯化α1AGP，用于小鼠免疫原。Western blot分析获得3个识别牛α - 1agp的单抗（IgM with κ轻链）。经Western blot检测，单抗与α1AGP裂解后不发生反应。牛l -选择素的cDNA克隆及其蛋白表达：克隆牛L-sel cDNA，其ORF为1110 bp，编码370个氨基酸。在细菌表达中，重组蛋白是不溶的。在杆状病毒-昆虫细胞表达中，该蛋白以膜蛋白形式表达，与特异性抗体发生反应。多糖链分析：α1AGP在正常健康状态下可分解为与ConA高亲和力的2个部分，在牛白血病中除了这2个部分外，还可分解为4个亲和力较低的部分。与E-sel一样，抗sialyl Lewis X抗体对牛α - 1agp的反应性较弱，但在牛白血病中似乎比在正常健康中更强。牛淋巴细胞功能分析：对牛淋巴细胞细胞因子mRNA进行分析。淋巴细胞中IL-2 mRNA的表达存在个体差异，但IL-4表达降低，IFN-γ表达升高，存在个体差异。

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.