Vanlandschoot P, Beirnaert E, Neirynck S, Saelens X, Jou W M, Fiers W
Laboratory of Molecular Biology, University of Ghent, Belgium.
Arch Virol. 1996;141(9):1715-26. doi: 10.1007/BF01718294.
A/Victoria/3/75 (H3N2)-derived cDNA coding for a secreted haemagglutinin (HA0s) was cloned into the polyhedrin promoter-based pVL1392 transfer vector, and a recombinant baculovirus was isolated. 5 to 10 micrograms/ml of secreted HA were obtained following infection of Spodoptera frugiperda-9 cells. Gel filtration revealed the presence in the cell supernatant of immunoreactive HA molecules with varying M(r). The high M(r) fraction (aHA0s) could be purified by Matrex Cellufine Sulphate and Lentil-lectin affinity chromatography, followed by Sephacryl S300 HR gel filtration. aHA0s consisted of aggregated, non-covalently linked subunits which were not cleaved into HA1 and HA2 polypeptides; aHA0s was highly susceptible to trypsin treatment and reacted with two low pH-specific monoclonal antibodies, suggesting that a HA0s consists of monomeric subunits. When the expression medium was adjusted to pH 8.5, no aHA0s was observed, suggesting that aggregation occurred in the cells due to a low intracellular pH. Balb/c mice immunized with purified aHA0s developed high, aHA0s-specific antibody titres. Despite these high titres, almost no binding to trimeric viral HA was observed, and immunized mice were not protected against a challenge with homologous mouse-adapted X47 virus. However, when virus was subjected to low pH, resulting in a profound conformational rearrangement, strong binding was observed. Moreover, binding to the low pH-treated HA of different drift variants, isolated between 1968 and 1989, occurred.
编码分泌型血凝素（HA0s）的源自A/维多利亚/3/75（H3N2）的互补DNA（cDNA）被克隆到基于多角体蛋白启动子的pVL1392转移载体中，并分离出重组杆状病毒。用草地贪夜蛾-9细胞感染后，获得了5至10微克/毫升的分泌型HA。凝胶过滤显示细胞上清液中存在具有不同相对分子质量（M(r)）的免疫反应性HA分子。高分子量部分（aHA0s）可通过Matrex Cellufine Sulphate和扁豆凝集素亲和层析纯化，随后进行Sephacryl S300 HR凝胶过滤。aHA0s由聚集的、非共价连接的亚基组成，这些亚基未被切割成HA1和HA2多肽；aHA0s对胰蛋白酶处理高度敏感，并与两种低pH特异性单克隆抗体发生反应，表明aHA0s由单体亚基组成。当将表达培养基的pH值调至8.5时，未观察到aHA0s，这表明由于细胞内pH值较低，聚集发生在细胞内。用纯化的aHA0s免疫的Balb/c小鼠产生了高的、aHA0s特异性抗体滴度。尽管有这些高滴度，但几乎未观察到与三聚体病毒HA的结合，并且免疫小鼠未受到同源小鼠适应株X47病毒攻击的保护。然而，当病毒受到低pH处理，导致深刻的构象重排时，观察到强烈的结合。此外，还发生了与1968年至1989年间分离的不同漂移变异株的低pH处理HA的结合。
