PDF(Pubmed)
Abstract：
UNASSIGNED: Orf, also known as contagious ecthyma (CE), is an acute, contagious zoonotic disease caused by the orf virus (ORFV). The F1L protein is a major immunodominant protein on the surface of ORFV and can induce the production of neutralizing antibodies.
UNASSIGNED: The prokaryotic expression system was used to produce the recombinant F1L protein of ORFV, which was subsequently purified and used to immunize mice. Positive hybridoma clones were screened using an indirect enzyme-linked immunosorbent assay (ELISA). The reactivity and specificity of the monoclonal antibody (mAb) were verified through Western blot and indirect immunofluorescence (IFA). The linear antigenic epitope specific to the mAb was identified through Western blot, using truncated F1L proteins expressed in eukaryotic cells. A multiple sequence alignment of the ORFV reference strains was performed to evaluate the degree of conservation of the identified epitope.
UNASSIGNED: After three rounds of subcloning, a mAb named Ba-F1L was produced. Ba-F1L was found to react with both the exogenously expressed F1L protein and the native F1L protein from ORFV-infected cells, as confirmed by Western blot and IFA. The mAb recognized the core epitope 103CKSTCPKEM111, which is highly conserved among various ORFV strains, as shown by homologous sequence alignment.
UNASSIGNED: The mAb produced in the present study can be used as a diagnostic reagent for detecting ORFV and as a basic tool for exploring the mechanisms of orf pathogenesis. In addition, the identified linear epitope may be valuable for the development of epitope-based vaccines.
UNASSIGNED: The prokaryotic expression system was used to produce the recombinant F1L protein of ORFV, which was subsequently purified and used to immunize mice. Positive hybridoma clones were screened using an indirect enzyme-linked immunosorbent assay (ELISA). The reactivity and specificity of the monoclonal antibody (mAb) were verified through Western blot and indirect immunofluorescence (IFA). The linear antigenic epitope specific to the mAb was identified through Western blot, using truncated F1L proteins expressed in eukaryotic cells. A multiple sequence alignment of the ORFV reference strains was performed to evaluate the degree of conservation of the identified epitope.
UNASSIGNED: After three rounds of subcloning, a mAb named Ba-F1L was produced. Ba-F1L was found to react with both the exogenously expressed F1L protein and the native F1L protein from ORFV-infected cells, as confirmed by Western blot and IFA. The mAb recognized the core epitope 103CKSTCPKEM111, which is highly conserved among various ORFV strains, as shown by homologous sequence alignment.
UNASSIGNED: The mAb produced in the present study can be used as a diagnostic reagent for detecting ORFV and as a basic tool for exploring the mechanisms of orf pathogenesis. In addition, the identified linear epitope may be valuable for the development of epitope-based vaccines.
摘要：
■Orf,也被称为传染性湿疹(CE)，是急性的，由orf病毒（ORFV）引起的传染性人畜共患疾病。F1L蛋白是ORFV表面的主要免疫显性蛋白,可以诱导中和抗体的产生。
■原核表达系统用于生产ORFV的重组F1L蛋白，随后纯化并用于免疫小鼠。使用间接酶联免疫吸附测定（ELISA）筛选阳性杂交瘤克隆。通过Western印迹和间接免疫荧光（IFA）验证了单克隆抗体（mAb）的反应性和特异性。通过Westernblot鉴定了单克隆抗体特异性的线性抗原表位。使用截短的F1L蛋白在真核细胞中表达。进行ORFV参考菌株的多序列比对以评估所鉴定的表位的保守程度。
■经过三轮亚克隆，产生名为Ba-F1L的mAb。发现Ba-F1L与外源表达的F1L蛋白和来自ORFV感染细胞的天然F1L蛋白反应，通过Westernblot和IFA证实。mAb识别核心表位103CKSTCPKEM111，在各种ORFV菌株中高度保守，如同源序列比对所示。
■本研究中产生的mAb可用作检测ORFV的诊断试剂，并可作为探索ORFV发病机理的基本工具。此外,鉴定的线性表位对于开发基于表位的疫苗可能是有价值的。
■原核表达系统用于生产ORFV的重组F1L蛋白，随后纯化并用于免疫小鼠。使用间接酶联免疫吸附测定（ELISA）筛选阳性杂交瘤克隆。通过Western印迹和间接免疫荧光（IFA）验证了单克隆抗体（mAb）的反应性和特异性。通过Westernblot鉴定了单克隆抗体特异性的线性抗原表位。使用截短的F1L蛋白在真核细胞中表达。进行ORFV参考菌株的多序列比对以评估所鉴定的表位的保守程度。
■经过三轮亚克隆，产生名为Ba-F1L的mAb。发现Ba-F1L与外源表达的F1L蛋白和来自ORFV感染细胞的天然F1L蛋白反应，通过Westernblot和IFA证实。mAb识别核心表位103CKSTCPKEM111，在各种ORFV菌株中高度保守，如同源序列比对所示。
■本研究中产生的mAb可用作检测ORFV的诊断试剂，并可作为探索ORFV发病机理的基本工具。此外,鉴定的线性表位对于开发基于表位的疫苗可能是有价值的。
