Vanderheijden N, De Moerlooze L, Vandenbergh D, Chappuis G, Renard A, Lecomte C
Eurogentec s.a., Parc de recherches de la Cense Rouge, Seraing, France.
J Gen Virol. 1993 Jul;74 ( Pt 7):1427-31. doi: 10.1099/0022-1317-74-7-1427.
The putative gene encoding the cytopathic bovine viral diarrhoea virus (BVDV) Osloss strain p80 protein was amplified by PCR and inserted into a T7 promoter-based vector for expression in Escherichia coli. Bacterial expression led to cytoplasmic insoluble inclusion bodies which were denatured by urea treatment and renatured by dialysis. Rabbit antisera were raised against this p80 recombinant antigen and assayed for the immunoprecipitation of either p120 or p80 protein from cytopathic or non-cytopathic BVDV biotype-infected bovine cells. The p80 gene sequence was also integrated into a baculovirus genome for its expression in Spodoptera frugiperda insect cells. The recombinant proteins isolated from bacteria or insect cells showed distinct antigenic properties when analysed by ELISA. Their ability to detect anti-BVDV specific antibodies was examined in a monoclonal antibody-based competitive ELISA performed on a series of field cattle sera. This comparative assay revealed the superiority of the insect cell-mediated expression to mimic the natural BVDV antigen produced by cell culture. The baculovirus/insect cell recombinant antigen gave the highest correlation between the ELISA-detected antibodies and the corresponding virus neutralization data.
通过聚合酶链反应（PCR）扩增了编码致细胞病变的牛病毒性腹泻病毒（BVDV）奥斯拉斯毒株p80蛋白的假定基因，并将其插入基于T7启动子的载体中，以便在大肠杆菌中表达。细菌表达产生了细胞质不溶性包涵体，经尿素处理使其变性，再通过透析使其复性。用该p80重组抗原制备兔抗血清，并检测其对致细胞病变或非致细胞病变BVDV生物型感染的牛细胞中p120或p80蛋白的免疫沉淀作用。p80基因序列也被整合到杆状病毒基因组中，以便在草地贪夜蛾昆虫细胞中表达。通过酶联免疫吸附测定（ELISA）分析，从细菌或昆虫细胞中分离出的重组蛋白显示出不同的抗原特性。在基于单克隆抗体的竞争性ELISA中，对一系列田间牛血清进行检测，以检验它们检测抗BVDV特异性抗体的能力。这种比较试验揭示了昆虫细胞介导的表达在模拟细胞培养产生的天然BVDV抗原方面的优越性。杆状病毒/昆虫细胞重组抗原在ELISA检测的抗体与相应的病毒中和数据之间具有最高的相关性。
