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目的 抗体在痘病毒的免疫预防中扮演着重要的作用，本研究对实验室分离的牛结节性皮肤病病毒（Lumpy skin disease virus,LSDV）毒株ORF122基因，采用生物信息学方法分析其编码蛋白的理化性质与结构特征。方法 以LSDV基因组DNA为模板扩增ORF122基因胞外区片段，构建到原核表达载体pET-30a中，利用IPTG诱导、表达、纯化目的蛋白，并免疫大白兔，制备多克隆抗体；通过Western blot、IFA、病毒中和试验对多克隆抗体特性进行测定。结果 构建的原核表达载体pET-30a-122b转化至Rosetta(DH3)表达菌后，IPTG诱导表达分子量约为19 kDa的蛋白，与预期分子量大小相符。Western blot分析表明表达的蛋白能被LSDV阳性血清识别，间接ELISA测定多克隆抗体效价1∶260416；免疫荧光结果进一步表明∶制备的多克隆抗体能够特异性识别LSDV感染羊睾丸细胞表达的天然蛋白，表明其具有良好的反应性，病毒中和试验表明该抗体能够中和病毒阻止LSDV感染。结论 本研究通过高效表达LSDV ORF122b蛋白，并分析其免疫原性、反应性及抗体中和活性，为深入研究痘病毒致病和免疫机理，进而快速建立其诊断方法，为牛结节性皮肤病防控技术措施的研究奠定基础。
Abstract：Objective Antibodies play an important role in the immunoprophylaxis of poxviruses. In this study, the ORF122 gene of a laboratory-isolated lumpy skin disease virus(LSDV) strain was analyzed using bioinformatics methods to characterize the physicochemical properties and structural features of its encoded protein. Methods The extracellular region of the ORF122 gene was amplified using LSDV genomic DNA as a template and cloned into the prokaryotic expression vector pET-30a. The recombinant protein was induced with IPTG, expressed and purified, and then used to immunize New Zealand white rabbits for the production of polyclonal antibodies. The characteristics of the polyclonal antibodies were evaluated by Western blot, indirect immunofluorescence assay(IFA), and virus neutralization test. Results The recombinant prokaryotic expression vector pET-30a-122b was successfully transformed into E. coli Rosetta(DH3). After IPTG induction, a protein of approximately 19 kDa was expressed, consistent with the predicted molecular weight. Western blot analysis showed that the expressed protein could be specifically recognized by LSDV-positive serum. The titer of the polyclonal antibody determined by indirect ELISA reached 1∶ 260416. Immunofluorescence assay further demonstrated that the prepared polyclonal antibody could specifically recognize the native protein expressed in LSDV-infected ovine testicular cells, indicating good reactivity. Virus neutralization assays showed that the antibody was able to neutralize LSDV and inhibit viral infection. Conclusion In this study, the LSDV ORF122b protein was efficiently expressed, and its immunogenicity, reactivity, and neutralizing activity were systematically analyzed. These findings provide a basis for further investigation of poxvirus pathogenesis and immune mechanisms, facilitate the rapid development of diagnostic methods, and lay a foundation for the prevention and control of lumpy skin disease.
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基本信息:
DOI：10.13242/j.cnki.bingduxuebao.250414
中图分类号:S858.23
引用信息:
[1]常慧慧,张慧,景伟,等.牛结节性皮肤病病毒ORF122编码蛋白多克隆抗体的制备及特性研究[J].病毒学报,2026,42(04):1140-1148.DOI:10.13242/j.cnki.bingduxuebao.250414.
基金信息:
“十四五”国家重点研发计划（项目号∶2023YFD1802501）,题目∶牛结节皮肤病灭活疫苗创制; 兰州市科技计划项目（项目号∶2023-1-38）,题目∶牛结节皮肤病两种试剂盒的创制、生产与示范应用; 国家自然科学基金面上项目（项目号∶32473069）,题目∶我国LSDV疫苗样重组流行毒株及其再重组与生物学特性研究; 甘肃省科技重大专项（项目号∶23ZDNA007）,题目∶动物疫病防控关键技术创新与重大产品研发~~
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