摘要
【目的】探究鸡早幼粒细胞白血病蛋白核小体(PML NBs)在马立克病病毒(Marek’s disease virus,MDV)感染过程中的生物学功能,制备抗鸡早幼粒细胞白血病蛋白(Ch-PML)单克隆抗体,建立可视化检测Ch-PML的方法。【方法】通过原核表达系统制备Ch-PML重组蛋白,并将其作为免疫原免疫BALB/c小鼠,采用细胞融合技术将骨髓瘤细胞和免疫小鼠的脾细胞进行融合,用ELISA方法筛选出分泌特异性抗体的杂交瘤细胞株,通过体内诱生法制备腹水并用Protein A/G纯化抗Ch-PML单克隆抗体,利用Western blotting和ELISA方法检测单克隆抗体的特异性、亲和力和亚类,最后利用该单克隆抗体建立用于鸡PML NBs的间接免疫荧光试验(IFA)检测方法。【结果】Ch-PML蛋白在大肠杆菌中可溶性表达,分子质量大小为19 ku;以此蛋白成功制备了1株抗Ch-PML单克隆抗体,该抗体具有良好的特异性和高亲和力,经亚类和型检测,其为IgG2b亚类,轻链为Kappa型。利用制备的抗Ch-PML单克隆抗体建立的IFA检测方法发现,内源性PML NBs在宿主细胞核中呈点状分布,过表达的Ch-PML在宿主细胞核中形成团块状聚集体,且MDV感染宿主细胞后核内PML NBs数量比未感染细胞显著减少(P<0.05),说明MDV感染导致核内的PML NBs组装受到抑制。【结论】本研究利用制备的抗Ch-PML单克隆抗体建立了鸡PML NBs的IFA检测方法,并发现MDV抑制宿主细胞PML NBs组装这一现象,为MDV致病机制的解析提供了新的研究思路和工具。
【Objective】This study was to explore the biological function of chicken promyelocytic leukemia protein nuclear bodies(PML NBs)in the process of Marek’s disease virus(MDV)infection,and establish a method to visualization and detection of chicken promyelocytic leukemia protein(Ch-PML)based on a monoclonal anti-Ch-PML.【Method】Recombinant Ch-PML protein was prepared by the prokaryotic expression system and was used as immunogen to immunize BALB/c mice.Myeloma cells and splenocytes of the immunized mice were fused by cell fusion technology,and a hybridoma cell line secreting specific antibodies anti-Ch-PML was screened by ELISA.The in vivo method for the production of ascites and the corresponding antibody specific anti-Ch-PML was obtained via purification with Protein A/G.Western blotting and ELISA were used to determine the specificity,affinity and subclass of the monoclonal antibody,and finally the monoclonal antibody was used to establish an indirect immunofluorescence assay(IFA)for the detection of chicken PML NBs.【Result】Ch-PML was expressed soluble in E.coli with a molecular weight of 19 ku.An anti-Ch-PML monoclonal antibody was successfully produced which had high specificity and affinity,and was detected by subclass and type as an IgG2b subclass with a Kappa-type light chain.An IFA was developed using the prepared anti-Ch-PML monoclonal antibody.The results showed that endogenous PML NBs were distributed in the nucleus of host cells in a punctate pattern,and overexpressed Ch-PML formed clumped aggregates in the nucleus of host cells.The number of PML NBs in the nucleus of MDV-infected cells was significantly reduced compared with that of uninfected cells(P<0.05),indicating that MDV infection led to the inhibition of PML NBs assembly in the nucleus.【Conclusion】In this study,a fluorescence detection method for chicken PML NBs was established by the prepared anti-Ch-PML monoclonal antibody,and discovered the phenomenon that MDV inhibited the assembly of PML NBs in host cells,which provided a ne
作者
曹梦瑶
王晶
周林宜
程晶
李永清
许健
江波
胡格
CAO Mengyao;WANG Jing;ZHOU Linyi;CHENG Jing;LI Yongqing;XU Jian;JIANG Bo;HU Ge(College of Animal Science and Technology,Beijing University of Agriculture,Beijing 102206,China;Institute of Animal Husbandry and Veterinary Medicine,Beijing Academy of Agricultural and Forestry Sciences,Beijing 100097,China;College of Veterinary Medicine,China Agricultural University,Beijing 100193,China;Lanzhou Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Lanzhou 730070,China)
出处
《中国畜牧兽医》
CAS
CSCD
北大核心
2024年第10期4500-4509,共10页
China Animal Husbandry & Veterinary Medicine
基金
国家自然科学基金青年基金项目(31902245)
北京市农林科学院科技创新能力建设专项(KJCX20220422)
北京市农林科学院畜牧兽医研究所改革与发展项目(XMS202403)。
