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PURIFICATION AND CHARACTERIZATION OF INFLUENZA A VIRUS INHIBITORS IN ANIMAL SERA

动物血清中甲型流感病毒抑制剂的纯化和表征

基本信息

批准号：
03670234
负责人：
WAKAMIYA Nobutaka
金额：
$ 1.28万
依托单位：
OSAKA UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1991
资助国家：
日本
起止时间：
1991 至 1992
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-03670234/
关键词：
INFLUENZA VIRUS INHIBITOR C-LECTIN インヒビタ-シアル酸 HanganutziuーDeicher(HD)抗原

项目摘要

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.

正常的马和豚鼠血清含有α 2-巨球蛋白，其抑制甲型流感病毒H2和H3亚型的感染性和血凝活性。另一方面，正常牛血清含有称为β抑制剂的组分，其抑制对H1和H3亚型甲型流感病毒的感染性和血凝活性。为了研究A型流感病毒β抑制剂的性质，我们纯化了连接蛋白并检测了其特性。从牛血清中纯化的连接蛋白对H1和H3亚型的A型流感病毒具有中和活性和HI活性。刀豆素的HI活性是热稳定的，Ca^++依赖的，并且对神经氨酸酶和高碘酸盐处理都有抗性。纯化后的刀豆素的HI活性可被N-乙酰氨基葡萄糖阻断，但不被D-甘露糖阻断。结合蛋白与血凝素结合，血凝素具有高甘露糖和复杂的糖链，其结合被N-乙酰氨基葡萄糖抑制，并依赖于低价阳离子。这些数据表明，牛血清的β-样抑制剂活性主要依赖于抑制血凝并通过与HA上的碳水化合物位点结合来中和病毒感染性的补体。其次，我们试图克隆编码牛conglutinin的cDNA。使用从PCR产物获得的特异性探针，从牛肝cDNA文库中分离编码牛连接蛋白的cDNA克隆。这些cDNA携带1113 bp的插入片段，编码371个氨基酸残基的蛋白质，其中20个残基为信号肽。对牛基因组DNA的Southern杂交分析表明，编码牛conglutinin的基因只有一个拷贝。牛组织的北方印迹分析表明，约1.5 kb的conglutinmRNA在肝中表达，在肺中也有少量表达。