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

点击翻译按钮获取中文摘要

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.