非洲猪瘟病毒p62蛋白单克隆抗体的制备及初步应用

为制备非洲猪瘟病毒（ASFV）p62蛋白的特异性单克隆抗体，并初步应用于感染组织样品中ASFV抗原的免疫组化（IHC）检测，本研究以杆状病毒表达的非洲猪瘟病毒重组p62蛋白免疫BALB/c小鼠，取其脾细胞与骨髓瘤细胞进行融合获得杂交瘤细胞。结果显示：基于纯化的p62蛋白建立的间接ELISA方法对杂交瘤细胞进行筛选和亚克隆，获得了18株可稳定分泌抗非洲猪瘟病毒p62蛋白单克隆抗体的杂交瘤细胞株。经IFA检测，制备的单克隆抗体均与非洲猪瘟病毒反应，且不与猪瘟病毒、猪繁殖与呼吸综合征病毒、猪伪狂犬病病毒、猪圆环病毒2型等猪源常见病毒反应，特异性良好。抗体识别蛋白的鉴定结果显示，3株MAbs识别p35蛋白，15株MAbs识别p15蛋白。14株MAbs重链亚类为IgG1型，4株MAbs重链亚类为IgG2a，轻链均为κ链。利用18株MAbs对ASFV感染猪的肺、扁桃体、淋巴结等组织进行IHC检测，结果显示5株MAbs均能够与感染ASFV的组织发生特异性的免疫反应。本研究获得的非洲猪瘟病毒p62蛋白单克隆抗体可为非洲猪瘟病毒免疫学检测方法的建立及p62蛋白的结构功能等基础研究提供重要的生物材料。

Preparation of Monoclonal Antibodies against Recombinant p62 Protein of African Swine Fever Virus and Its Preliminary Application

The objective of this study was to prepare monoclonal antibodies against p62 protein of African swine fever virus (ASFV), and apply it to immunohistochemistry (IHC) detection of ASFV antigen in infected tissue samples. BALB/c mice were immunized with purified recombinant p62 protein expressed by baculovirus system and hybridoma cells were obtained by fusion of spleen cells and myeloma cells. An indirect ELISA based on the purified p62 protein was developed. Eighteen hybridoma cell lines stably secreting MAbs against the p62 protein were harvested after screening and subcloning. The results of indirect immunofluorescence assay (IFA) showed that all of the monoclonal antibodies could react with ASFV and did not react with swine fever virus, porcine reproductive and respiratory syndrome virus, porcine pseudorabies virus and porcine circovirus 2 with good specificity. Three MAbs could recognize ASFV p35 protein, and 15 MAbs recognize p15 protein. Among of them, 14 MAbs heavy chains belong to IgG1 and 4 MAbs heavy chains belong to IgG2a. All light chains were κ. The results of immunohistochemistry showed that 5 MAbs had positive reactivity with the ASFV in lung, tonsil, lymph node of pigs infected with ASFV. The monoclonal antibodies against p62 protein obtained from the study laid a foundation for the establishment of immunological detection method of African swine fever virus and could provide important biomaterials for research on the structure and function of p62 protein.