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为制备猪盖塔病毒（GETV）Cap蛋白多克隆抗体并探索其应用潜力，首先构建pCold-TF-Cap重组质粒，通过0.4 mmol/L IPTG诱导高效表达可溶性TF-Cap融合蛋白。经Ni-NTA亲和层析纯化后，利用SDS-PAGE鉴定重组蛋白纯度较高，BCA法测得蛋白浓度为22 mg/m L。以纯化的融合蛋白与佐剂乳化后免疫6～8周龄BALB/c小鼠，成功制备效价达1∶128 000的鼠源多克隆抗体。通过Western-blot分析与间接免疫荧光试验（IFA），证实该多克隆抗体能够特异性识别GETV感染的BHK-21细胞内源性Cap蛋白。此外，以制备的多克隆抗体为一抗、酶标羊抗鼠Ig G为二抗，建立了检测GETV感染小鼠脾和肝组织中Cap蛋白的免疫组织化学（IHC）检测方法。结果显示，阳性信号清晰定位于细胞质中，呈现明显的棕色信号。本研究成功制备了特异性强的GETV Cap蛋白多克隆抗体，并建立了配套IHC检测方法，为GETV的基础研究和临床诊断提供了可靠的免疫学工具。
Abstract：To prepare polyclonal antibodies against the capsid(Cap) protein of porcine Getah virus(GETV) and explore their potential applications,the recombinant plasmid p Cold-TF-Cap was constructed in this study. Soluble TF-Cap fusion protein was efficiently expressed by induction with 0.4 mmol/L isopropyl-β-D-thiogalactopyranoside(IPTG).After purification by Ni-NTA affinity chromatography,the recombinant protein exhibited high purity as verified by SDS-PAGE,with a protein concentration of 22 mg/m L as determined by the BCA assay. Subsequently,BALB/c mice(aged 6—8 weeks) were immunized with purified fusion protein emulsified in adjuvant,resulting in mouse polyclonal antibodies with a high titer of up to 1 ∶ 128 000. Western-blot analysis and indirect immunofluorescence assay(IFA) demonstrated that the prepared polyclonal antibodies specifically recognized endogenous GETV Cap protein in infected BHK-21 cells. In addition,an immunohistochemistry(IHC) detection method was established using the prepared polyclonal antibody as the primary antibody and HRP-conjugated goat anti-mouse Ig G as the secondary antibody,enabling the detection of Cap protein in spleen and liver tissues of GETV-infected mice. The results revealed clear positive signals localized within the cytoplasm,characterized by distinct brown staining.In conclusion,this study successfully generated a highly specific polyclonal antibody against GETV Cap protein and developed a reliable IHC detection method,providing valuable immunological tools for both basic research and clinical diagnosis of GETV infection.
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基本信息:
DOI：10.16656/j.issn.1673-4696.2025.0236
中图分类号:S852.651
引用信息:
[1]魏炳燕,邢璐璐,张璞,等.猪盖塔病毒Cap蛋白的原核表达及多克隆抗体的制备[J].中国兽医科学,2026,56(01):83-89.DOI:10.16656/j.issn.1673-4696.2025.0236.
基金信息:
国家重点研发计划项目（2024YFD1800200）; 中国农业大学2115人才工程项目
2025-08-05
2025-08-05
2025-08-05