Härkönen T, Hovi T, Roivainen M
Enterovirus Laboratory, National Public Health Institute, Helsinki, Finland.
J Virol Methods. 1997 Dec;69(1-2):147-58. doi: 10.1016/s0166-0934(97)00150-x.
Coxsackievirus B4 (CBV-4) capsid protein VP0 and non-structural 2C protein were expressed and purified using a glutathione-S-transferase (GST) fusion protein expression system. We used a full-size CBV-4 cDNA as a template to amplify the genes by polymerase chain reaction (PCR). The genes were cloned into expression vector pGEX-2T and expressed as a fusion protein with GST. The GST-fusion proteins (GST-2C and GST-VP0) were purified in denatured and native forms and used to generate antibodies in rabbits. The antisera raised against GST-VP0 fusion protein recognized the corresponding structural proteins (VP0, VP2 and VP4) from purified CBV-4 preparations and infected cell lysates. In addition, cross-reactivity with CAV-9 and CBV-5 capsid proteins was observed. Anti-GST-2C antisera precipitated viral 2C protein in CBV-4-infected GMK cells, showing that the antibodies recognize the corresponding natural antigen.
柯萨奇病毒B4（CBV - 4）衣壳蛋白VP0和非结构蛋白2C通过谷胱甘肽 - S - 转移酶（GST）融合蛋白表达系统进行表达和纯化。我们以全长CBV - 4 cDNA为模板，通过聚合酶链反应（PCR）扩增基因。将这些基因克隆到表达载体pGEX - 2T中，并作为与GST的融合蛋白进行表达。GST融合蛋白（GST - 2C和GST - VP0）以变性和天然形式纯化，并用于在兔体内产生抗体。针对GST - VP0融合蛋白产生的抗血清识别来自纯化的CBV - 4制剂和感染细胞裂解物中的相应结构蛋白（VP0、VP2和VP4）。此外，还观察到与CAV - 9和CBV - 5衣壳蛋白的交叉反应性。抗GST - 2C抗血清在CBV - 4感染的GMK细胞中沉淀出病毒2C蛋白，表明这些抗体识别相应的天然抗原。
