Crystalline antigen from the influenza virus envelope.
Crystalline antigen from the influenza virus envelope.
复制标题
来自流感病毒包膜的晶体抗原。
DOI：
--
复制
发表时间：
1972
期刊：
影响因子：
--
通讯作者：
J. Skehel
中科院分区：
文献类型：
--
作者：
C. Brand;J. Skehel
流感病毒的包膜含有两种形态和化学上不同的糖蛋白，一种是血凝素-TiNiN，另一种是神经氨酸酶，最近的实验表明，用菠萝酶处理病毒粒子可以将其去除。在对这一现象机理的研究中发现，尽管神经氨酸酶蛋白在蛋白降解过程中被完全降解，但分离的血凝素蛋白可以从孵育混合物中几乎不变地恢复。我们报道了随后的血凝素的纯化和结晶，以及它的初步化学和抗原性表征。所描述的大多数实验中都使用了A2/Hong Kong/682的X-31菌株。病毒在胚胎鸡蛋中生长，按照Skehel和SChild3的描述进行纯化，并按照Compans等人的描述用菠萝酶消化。100,000g离心60min，蔗糖密度梯度离心法(20-60%蔗糖，磷酸盐缓冲盐水，100,000g，16h)纯化病毒颗粒。与完整病毒相比，这些颗粒的形态如图1所示。很明显，在蛋白质消化过程中，所有的包膜尖峰蛋白都被去除了；与这些观察结果一致，剃除的颗粒完全没有病毒包膜蛋白的血凝和神经氨酸酶活性。对这些颗粒的多肽组成的聚丙烯酰胺凝胶电泳分析也表明，它们只含有病毒4的7个多肽中的4个，其比例与完整的病毒颗粒中存在的相同(图2B和C)。在酶处理过程中，血凝活性和神经氨酸酶活性均被完全破坏。然而，对去除亚病毒颗粒后的蛋白酶孵育上清液中存在的蛋白质的电泳分析表明，这种混合物的主要成分是两种流动性类似于先前鉴定的血凝素多肽的多肽3。因此，上清液中较大的多肽的迁移率与较大的血凝素组分的迁移率相同，而较小的多肽的迁移率表明该上清液的较大的多肽与较小的血凝素多肽的相对分子质量相差约3,000。
THE envelope of influenza viruses contains two morphologically and chemically distinct glycoproteins, a haemagglu-tinin and a neuraminidase, which recent experiments have indicated1 may be removed from the virion by treatment with the protease, bromelain. In an investigation of the mechanism of this phenomenon it was found that although the neuraminidase protein was completely degraded during proteolysis, the isolated haemagglutinin protein could be recovered almost unchanged from the incubation mixture. We report the subsequent purification and crystallization of the haemagglutinin and its preliminary chemical and antigenic characterization. The X-31 strain of A2/Hong Kong/682 was used in most of the experiments to be described. Viruses were grown in embryonated eggs, purified as described by Skehel and Schild3 and digested with bromelain as described by Compans et al.1. The smooth-surfaced virus particles which resulted were removed from the incubation mixture by centrifuging for 60 min at 100,000g and purified by sucrose density gradient centrifugation (20–60% sucrose in phosphate buffered saline (PBS) (100,000g, 16 h). The morphology of these particles in comparison with intact virus is shown in Fig. 1. It is clear that all of the envelope spike proteins were removed during proteolytic digestion; in agreement with these observations the shaved particles were completely devoid of the haemagglutinating and neuraminidase activities of the virus envelope proteins. Polyacrylamide gel electrophoretic analyses of the polypeptide composition of the particles also indicated that they contained only four of the seven polypeptides of the virus4 in the same proportions as these were present in the intact virus particles (Fig. 2b and c). Both haemagglu-tination and neuraminidase activities were completely destroyed during protease treatment. Electrophoretic analyses of the proteins present in the protease incubation supernatant after removal of the subviral particles indicated, however (Fig. 2a), that the major components of this mixture were two polypeptides of similar mobility to the haemagglutinin polypeptides previously identified3. Thus, the mobility of the larger polypeptide of the supernatant was identical to that of the larger haemagglutinin component and the mobility of the smaller polypeptide indicated a molecular weight difference of approximately 3,000 between this and the smaller haemagglutinin polypeptide.