PURIFICATION AND CHARACTERIZATION OF INFLUENZA A VIRUS INHIBITORS IN ANIMAL SERA

正常的马和豚鼠血清中含有抑制H2和H3亚型甲型流感病毒的传染性和血凝活性的a2-巨球蛋白。另一方面，正常牛血清含有一种称为β抑制剂的成分，可抑制H1和H3亚型甲型流感病毒的传染性和血凝活性。为了研究甲型流感病毒β -抑制剂的性质，我们纯化了凝集素并检测了其特性。从牛血清中纯化的凝集素对H1和H3亚型甲型流感病毒具有中和活性和HI活性。粘连素的HI活性是热稳定的，依赖于Ca^<++>，并且对神经氨酸酶和高碘酸盐处理都有抗性。纯化后的凝集素的HI活性可被n -乙酰氨基葡萄糖抑制，而不被D-甘露糖抑制。凝集素与具有高甘露糖和复杂糖链的血凝素结合，其结合被N-乙酰氨基葡萄糖抑制，依赖于降价阳离子。这些数据表明，牛血清β样抑制剂的活性主要依赖于凝集素，凝集素通过与HA上的碳水化合物位点结合来抑制血凝并中和病毒的感染性。接下来，我们尝试克隆编码牛粘连素的cDNA。从牛肝脏cDNA文库中分离到编码牛凝集素的cDNA克隆。这些cdna携带一个1113 bp的插入，编码一个371个氨基酸残基的蛋白质和一个20个残基的信号肽。牛基因组DNA的Southern blot分析表明，编码牛粘连素的基因只有一个拷贝。牛组织的Northern blot分析显示，凝集素mRNA约1.5 kb在肝脏中表达，在肺中也有少量表达。

Normal horse and guinea pig sera contain a2-macroglobulin which inhibits the infectivity and hemagglutinating activity of influenza A viruses of the H2 and H3 subtypes. On the other hand, normal bovine serum contains a component termed beta inhibitor that inhibits the infectivity and hemagglutinating activity on influenza A viruses of the H1 and H3 subtypes. To investigate the nature of the beta inhibitor of influenza A virus, we purified the conglutinin and examined its characteristics. The conglutinin , purified from bovine serum, had neutralizing-activity as well as HI activity on influenza A viruses of the H1 and H3 subtypes. The HI activity of conglutinin was heat stable, Ca^<++>-dependent, and resistant to both neuraminidase and periodate treatments. The HI activity of purified conglutinin was blocked by N-acetylglucosamine but not by D- mannose. The conglutinin was bound to hemagglutinin which had high mannose and complex sugar chains and its binding was inhibited by N- acetylglucosamine and dependent on devalent cations. These data indicate that the beta-like inhibitor activity of bovine serum is mainly dependent on conglutinin which inhibits hemagglutination and neutralizes the virus infectivity by its binding to a carbohydrate site at the HA. Nextly, we tried to clone the cDNA coding for bovine conglutinin. cDNA clones encoding the bovine conglutinin were isolated from a bovine liver cDNA library using a specific probe obtained from the PCR product. These cDNAs carry an insert of 1113 bp coding for a protein of 371 amino acid residues with a signal peptide of 20 residues. Southern blot analysis of total bovine genomic DNA indicated that there is only one copy of the gene encoding bovine conglutinin. Northern blot analysis of bovine tissues showed that conglutinin mRNA of about 1.5 kb is expressed in the liver and also slightly in the lung.