Masri S A, Nagata L, Mah D C, Lee P W
Virology. 1986 Feb;149(1):83-90. doi: 10.1016/0042-6822(86)90089-9.
A cDNA clone encompassing the complete reovirus (serotype 3) S1 gene was constructed using two partial clones containing overlapping sequences. The gene (with the first 15 bases at the 5' end up to and including the first ATG removed) was then inserted in frame into the lac cloning site of the pUC13 plasmid and expressed in Escherichia coli as a fusion product under control of the lac promoter. The expressed product can be immunoprecipitated as a 47,000-mol wt (47K) protein using several monoclonal anti-sigma 1 antibodies. Like authentic soluble sigma 1 from reovirus-infected cells, the expressed protein is capable of attaching to mammalian cells (mouse L fibroblasts) in a specific manner and of competing with reovirus particles for cell surface receptors. Lysates prepared from the recombinant plasmid-transformed, but not those from pUC13-transformed E. coli cells, were also found to exhibit hemagglutinating (HA) activity. Such hemagglutination was inhibited by a monoclonal anti-sigma 1 antibody previously shown to inhibit reovirus HA activity. It is concluded that both the host cell attachment domain and the hemagglutination domain on the expressed protein are functionally intact.
利用两个含有重叠序列的部分克隆构建了一个包含呼肠孤病毒（3型）S1基因完整序列的cDNA克隆。然后将该基因（去除5'端前15个碱基直至并包括第一个ATG）读框插入pUC13质粒的lac克隆位点，并在大肠杆菌中作为融合产物在lac启动子的控制下表达。使用几种抗σ1单克隆抗体，表达产物可作为一种47,000分子量（47K）的蛋白质进行免疫沉淀。与呼肠孤病毒感染细胞中天然的可溶性σ1一样，表达的蛋白质能够以特定方式附着于哺乳动物细胞（小鼠L成纤维细胞），并与呼肠孤病毒颗粒竞争细胞表面受体。还发现，由重组质粒转化的大肠杆菌细胞制备的裂解物具有血凝（HA）活性，而pUC13转化的大肠杆菌细胞制备的裂解物则没有。先前已证明一种抗σ1单克隆抗体可抑制呼肠孤病毒HA活性，这种血凝反应也受到该抗体的抑制。得出的结论是，表达蛋白上的宿主细胞附着结构域和血凝结构域在功能上都是完整的。
