抗鸡早幼粒细胞白血病蛋白单克隆抗体的制备及其在荧光检测中的应用

doi:10.16431/j.cnki.1671-7236.2024.10.030

Abstract

Abstract: 【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 new research idea and tool for elucidating the pathogenic mechanism of MDV.

Key words: promyelocytic leukemia protein (PML); monoclonal antibody; NBs; Marek’s disease virus (MDV)

CLC Number:

CAO Mengyao, WANG Jing, ZHOU Linyi, CHENG Jing, LI Yongqing, XU Jian, JIANG Bo, HU Ge. Preparation of Monoclonal Antibody Anti-chicken PML and Its Application in Fluorescence Detection[J]. China Animal Husbandry and Veterinary Medicine, 2024, 51(10): 4500-4509.

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Preparation of Monoclonal Antibody Anti-chicken PML and Its Application in Fluorescence Detection

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