Russell George C, Percival Ann, Grant Dawn M, Bartley Kathryn, Turnbull Dylan, McLean Kevin, Lienhard Julia, Bachofen Claudia
Moredun Research Institute, Pentlands Science Park, Midlothian, UK.
Moredun Research Institute, Pentlands Science Park, Midlothian, UK.
J Virol Methods. 2022 Jan;299:114329. doi: 10.1016/j.jviromet.2021.114329. Epub 2021 Oct 12.
The minor capsid protein of ovine herpesvirus 2, identified as a potential antigen for serological testing, was over-expressed and purified to allow its assessment in ELISA. The corresponding gene sequence (OvHV-2 orf65, Ov65) was modified to incorporate epitope tags and internal restriction enzyme sites in an E. coli codon-optimised version of the gene. This codon-optimised gene was then subject to internal deletions to identify regions of the protein that could be removed while maintaining protein solubility and antigenicity. It was found that a derivative with deletion of the conserved 5'-end of the gene (Ov65delB) expressed a polypeptide that was soluble when over-expressed in bacteria and was detected by OvHV-2 specific sera. Proteomic analysis of the affinity purified Ov65delB showed that it contained multiple predicted Ov65 tryptic peptides but also showed contamination by co-purifying E. coli proteins. An indirect ELISA, based on this affinity-purified OV65delB, was optimised for use with sheep and cattle samples and cut-off values were established based on known negative serum samples. Analysis of groups of samples that were either presumed infected (UK sheep) or tested OvHV-2 positive or negative by PCR (cattle MCF diagnostic samples) showed that the assay had 95 % sensitivity and 96 % specificity for sheep serum; and 80 % sensitivity and 95 % specificity for cattle serum. The lower sensitivity with cattle samples appeared to be due to a lack of serological response in some MCF-affected cattle. This recombinant antigen therefore shows promise as the basis of an inexpensive, simple and reliable test that can be used to detect OvHV-2-specific antibody responses in both MCF-affected animals and in OvHV-2 reservoir hosts.
绵羊疱疹病毒2的次要衣壳蛋白被鉴定为血清学检测的潜在抗原，对其进行了过表达和纯化，以便在酶联免疫吸附测定（ELISA）中进行评估。对相应的基因序列（绵羊疱疹病毒2开放阅读框65，Ov65）进行修饰，在该基因的大肠杆菌密码子优化版本中加入表位标签和内部限制性酶切位点。然后对这个密码子优化的基因进行内部缺失，以确定蛋白质中在保持蛋白质溶解性和抗原性的同时可以去除的区域。结果发现，一种缺失基因保守5′端的衍生物（Ov65delB）表达的多肽在细菌中过表达时是可溶的，并且能被绵羊疱疹病毒2特异性血清检测到。对亲和纯化的Ov65delB进行蛋白质组学分析表明，它包含多个预测的Ov65胰蛋白酶肽段，但也显示出被共纯化的大肠杆菌蛋白污染。基于这种亲和纯化的OV65delB建立了一种间接ELISA，对其进行优化以用于绵羊和牛的样本，并根据已知的阴性血清样本确定了临界值。对假定感染（英国绵羊）或通过聚合酶链反应（PCR）检测为绵羊疱疹病毒2阳性或阴性的牛样本组（牛传染性单核细胞增多症诊断样本）进行分析表明，该检测方法对绵羊血清的灵敏度为95%，特异性为96%；对牛血清的灵敏度为80%，特异性为95%。牛样本较低的灵敏度似乎是由于一些受传染性单核细胞增多症影响的牛缺乏血清学反应。因此，这种重组抗原有望作为一种廉价、简单且可靠的检测方法的基础，可用于检测受传染性单核细胞增多症影响的动物和绵羊疱疹病毒2储存宿主中的绵羊疱疹病毒2特异性抗体反应。
