As the major ASFV envelope protein, p72 is one of the first viral proteins to induce the antibody production after viral infection and is an important marker in ASFV analysis and vaccine studies (Nan Wang et al. pB602L and validated its immunogenicity in serum from naturally infected pigs with ASFV. Furthermore, we successfully generated an IgG2a subclass monoclonal antibody (mAb 7E7) against pB602L using hybridoma technology. Using western blot and immunofluorescence assays, mAb 7E7 specifically identified the ASFV Pig/HLJ/2018/strain and eukaryotic recombinant ASFV pB602L protein in vitro. The 474SKENLTPDE482 epitope in the ASFV pB602L C-terminus was identified as the minimal linear epitope for mAb 7E7 binding, with dozens of truncated pB602l fragments characterized by western blot assay. We also showed that this antigenic epitope sequence has a high conservation and antigenic index. Our study contributes to improved vaccine and antiviral development and provides fresh insights into the serologic analysis of ASF. Key points ? genus (Alonso et al. 2018). The disease has a mortality rate of up to 100% and spreads rapidly due to its significant health and socioeconomic impact on international trade in pigs and pork products, and is a notifiable disease to the World Organization for Animal Health (WOAH) (Ward et al. 2021). The emergence of attenuated strains of ASFV in many countries and their quick spread pose severe difficulties for ASFV prevention and control actions in home and crazy pigs (Lv et al. 2021). However, the emergence of genotype I and recombinant strain variants offers improved transmission and mortality rates, and regrettably, a live attenuated vaccine derived from the ASFV genotype does not provide safety against recombinant viral assault (Zhao et al. 2023). Therefore, the emergence of naturally happening recombinant ASFV strains may present fresh difficulties to the global swine market. Overall, the global spread of ASFV is definitely highly complex and has severe implications for the global swine market and related economies (Forth et al. 2023; Kitamura et al. 2023). Vaccine development or treatment options for ASFV have been hampered by viral genome difficulty, insufficient info on host-virus relationships, and the many unidentified genes in the genome (Mu?oz-Prez et al. 2021; Wu et al. 2021; Zheng et al. 2022). In the absence of a vaccine, early analysis is definitely a critical prevention strategy. pB602L is definitely a late-stage non-structural protein that is not part of the viral particle, but is definitely a highly immunogenic protein of ASFV that may be used to develop diagnostic tools (Epifano et al. 2006). Like a molecular chaperone, it participates in the folding of the p72 nucleocapsid protein and is located outside the disease factory. The formation of the p72 trimeric structure is dependent within the co-expression of pB602L (Cobbold et al. 2001; Liu et al. 2019). Importantly, the absence of pB602L from your genome results in an irregular zipper-like structure that is unable to form icosahedral virus particles (Epifano et al. 2006). Earlier studies possess reported that pB602L elicits strong and specific reactions to the serum from convalescent ASFV (Gallardo et al. 2009; Gutirrez-Casta?eda et al. 2008). In addition, pB602L can be used to distinguish pigs which are naturally Cediranib maleate infected with crazy ASFV strains from pigs immunized with structural protein subunit vaccines (Gutirrez-Casta?eda et al. 2008; Irusta et al. 1996). Immunoglobulin type G (IgG) monoclonal antibodies (mAbs) are important reagents utilized for diagnostic and restorative purposes. However, the molecular basis of pB602L antigenicity and the mechanism behind its effects on p72 remains unclear. To day, very few studies have thus far generated specific mAbs against the pB602L protein and mapped its epitopes, limiting basic research on pB602L. To better understand the biological functions of ASFV pB602L, we used purified recombinant HIS-tagged pB602L protein and tested its ability to induce B-cell immunogenicity in pig serum after natural illness with ASFV. Using hybridoma technology, we generated mAb 7E7 that specifically bound to the ASFV Pig/HLJ/2018 strain. Due to the high specificity between both reagents, we recognized a B-cell linear epitope (474SKENLTPDE482) identified by mAb 7E7. Additionally, homology analyses Cediranib maleate showed that B-cell epitope sequences were highly conserved in several ASFV strains, including I, II, I and II recombinant, VIII, X, and XI genotype isolates. Our molecular docking approach showed that contact residues between 474SKENLTPDE482 and mAb 7E7 created various relationships, including salt bridges, hydrogen bonds, and hydrophobic relationships, which enhanced the affinity of Cediranib maleate the antibody to an antigen. Therefore, we offered fundamental insights and practical applications to ASFV. Materials and methods Serum samples Healthy swine-negative serum and standard positive sera for ASFV were from the China Veterinary Tradition Cediranib maleate Collection Center (Beijing, China). ASFV pB602L recombinant cloning, manifestation, and purification and detection of pB602L-specific antibodies in porcine serum samples Specific primers 5-TGGTGGTGCTCGAGTGCGGCCGCCAATTCTGCTTT-3 (F) and 5-ATGGGTCGCGGATCCGAATTCATGGCAGAATTTAA-3 (R) were designed based on the published ASFV gene sequence of ASFV (GenBank MK333180.1). Primers and genes were synthesized by Tsingke Biotechnology Co., Ltd. (Beijing, China). The amplified ASFV DNA Rabbit polyclonal to Aquaporin3 was subcloned into the expression vector pET-28a (+) Cediranib maleate (Novagen, San Diego, CA, USA) using BL21 (DE3) TSsetta.
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