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目的 制备针对2型人博卡病毒(Human bocavirus, HBoVs)VP3蛋白DR2多肽的特异性单克隆抗体(Monoclonal antibody,mAb)，并系统鉴定其特异性、亲和力。方法 针对HBoV2与HBoV1 VP3蛋白差异最显著的DR2区，合成特异性多肽并免疫BALB/c小鼠。通过细胞融合、杂交瘤筛选法，获得稳定分泌抗HBoV2-DR2单克隆抗体的杂交瘤细胞株。采用酶联免疫吸附(Enzyme linked immunosorbent assay,ELISA)、间接免疫荧光(Indirect immunofluorescence assay,IFA)鉴定抗体的特异性；利用生物膜层干涉技术(Bio-layer interferometry,BLI)测定抗体亲和力常数(KD)。结果 成功获得一株稳定分泌抗HBoV2-DR2 mAb的杂交瘤细胞株，命名为3A6，亚类鉴定为IgG1/κ型。ELISA和IFA结果显示，mAb 3A6与原核表达的HBoV2 VP3及表达HBoV2 VP3的细胞呈强阳性反应，而与HBoV1 VP3仅存在弱反应，与表达HBoV1 VP3的细胞未见阳性反应；在BLI检测中，其与HBoV2 VP3亲和力高，KD值达2.09×10-10M，与HBoV1 VP3未见显著结合，无KD值；提示3A6的区别于HBoV1的HBoV2型特异性。结论 本研究成功制备了一株具有高亲和力的抗HBoV2 DR2区的单克隆抗体，为HBoV2诊断试剂及药物研发奠定基础。
Abstract：Objective To generate a monoclonal antibody(mAb) specifically targeting the DR2 region of the VP3 protein of human bocavirus type 2(HBoV2) and to characterize its specificity and binding affinity. Methods The DR2 region, which shows the greatest sequence divergence between HBoV2 and HBoV1 VP3 proteins, was synthesized as a peptide and used to immunize BALB/c mice. Hybridoma cell lines were generated through cell fusion and screened to obtain clones stably secreting anti-HBoV2 DR2 mAb. The specificity and reactivity of the mAb were evaluated by enzyme-linked immunosorbent assay(ELISA) and indirect immunofluorescence assay(IFA). Binding affinity was determined using bio-layer interferometry(BLI) to calculate the equilibrium dissociation constant(KD). Results A hybridoma cell line stably secreting an antiHBoV2 DR2 mAb was successfully established and designated 3A6. Isotyping identified 3A6 as IgG1/κ. ELISA and IFA demonstrated strong reactivity of 3A6 with recombinant HBoV2 VP3 protein and cells expressing HBoV2 VP3. Only weak reactivity was observed with recombinant HBoV1 VP3, and no positive signal was detected in cells expressing HBoV1 VP3. BLI analysis showed that 3A6 bound to HBoV2 VP3 with high affinity, with a KD value of 2.09 × 10-10 M, whereas no significant binding to HBoV1 VP3 was detected. These results indicate that 3A6 exhibits high HBoV2 type specificity. Conclusion A high-affinity monoclonal antibody targeting the DR2 region of HBoV2 VP3 was successfully generated and characterized. This mAb provides a useful tool for the development of diagnostic reagents and therapeutic strategies against HBoV2.
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基本信息:
DOI：10.13242/j.cnki.bingduxuebao.250381
中图分类号:R373
引用信息:
[1]德日,张可祥,傅涵浩钰,等.靶向2型人博卡病毒DR2多肽的单克隆抗体制备及表征分析[J].病毒学报,2026,42(03):905-911.DOI:10.13242/j.cnki.bingduxuebao.250381.
基金信息:
国家自然科学基金（项目号：82502717）,题目：HBoV1儿童气道类器官分离培养模型的建立及关键宿主因子的筛选与鉴定;国家自然科学基金面上项目（项目号：82172277）,题目：人2型博卡病毒环状基因组的形成机制及其临床意义研究; 北京市自然科学基金资助项目（项目号：7254305）,题目：DDR通路通过HBoV1NS1蛋白关键结构域启动基因组环化机制研究; 北京市卫生健康委员会高层次公共卫生技术人才建设项目（项目号：学科带头人-02-20）,题目：儿童重症呼吸道感染病毒病原谱变迁及新发病毒病原预警体系建立~~
2026-04-23
2026-04-23
2026-04-23