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Chromogenic LAL Endotoxin Assay Kit ensures precise, FDA-compliant endotoxin quantification for biosafety testing.
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Powerful Tn5 Transposase for DNA insertion and random library construction.
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Aptamers for key proteins like ACVR1A, Akt, EGFR, and VEGFR.
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Monkeypox is a zoonotic viral disease caused by the monkeypox virus (MPXV), a member of the Orthopoxvirus genus, which also includes variola virus, cowpox virus, and vaccinia virus (VACV). Identified as a significant global health concern, monkeypox has seen increased attention due to rising incidence rates. The disease manifests with symptoms such as fever, rash, myalgia, and lymphadenopathy, resembling smallpox but typically presenting with milder symptoms. However, in immunocompromised individuals, monkeypox can be severe or fatal. MPXV is a linear double-stranded DNA virus with a genome of approximately 197 kb and shares a high degree of genetic similarity with other poxviruses.
Cell Culture: DMEM, Opti-MEM, FBS
Transfection Reagents: PEI
Antibodies: MPXV B6R and M1R polyclonal antibodies; MPXV A29L polyclonal antibody; Anti-His Tag monoclonal antibody; HRP-conjugated anti-mouse IgG, anti-rabbit IgG
Enzymes and Stains: PNGase F enzyme; Coomassie Brilliant Blue Fast Staining Solution; BCA Protein Quantification Kit
Instruments: Nucleic acid electrophoresis system, SDS-PAGE electrophoresis system, semi-dry transfer apparatus; HisTrap excel chromatography column, HiTrap Capto Q anion exchange column, AKTA purification system
Cells: HEK293T cells cultured in DMEM with 10% FBS at 37°C, 5% CO2.
Plasmids: MPXV A29L, B6R, and M1R genes synthesized with Kozak sequences and C-terminal His tags. Expression vectors used: pVRC-A29L, pVRC-B6R, pVRC-M1R, and pVRC empty vector.
Serum Samples: Neutralizing antibody titers against VACV and MPXV were measured by plaque reduction neutralization tests, including sera from mice, monkeys, and humans, as well as negative control sera.
Transfection: HEK293T cells were transfected with pVRC-A29L, pVRC-B6R, pVRC-M1R, and pVRC empty vector using PEI. Supernatants and cell lysates were collected 72 hours post-transfection.
Figure 1. Construction and Identification of Eukaryotic Expression Plasmids for MPXV A29L, B6R, and M1R Proteins (SP: tPA-1 signal peptide: MDAMKRGLCCVLLLCGAVFVSP).
Partner with Creative Biogene to ensure excellence in your monkeypox research and development. Discover more about our innovative solutions at Creative Biogene.
Protein Purification: Supernatants were filtered and proteins purified using HisTrap excel and HiTrap Capto Q columns. SDS-PAGE and Western blot verified purity and specificity. Protein concentrations were determined using the BCA method.
Procedure: A29L, B6R, and M1R proteins (100 ng/well) were coated on ELISA plates. Serum samples were diluted starting at 1:50 up to 1:819,200—secondary antibodies (anti-mouse IgG-HRP, anti-monkey IgG-HRP, anti-human IgG-HRP). Plates were developed with substrate and OD values were read using a plate reader.
Serum samples were serially diluted and mixed with MPXV or VTT virus. After 1 hour of incubation, virus-serum mixtures were added to cells for 1 hour, followed by the addition of a maintenance medium. Cells were incubated at 37°C with 5% CO2 for 72-96 hours. Plaque counts were assessed, with negative controls set to 10.
Data were analyzed using GraphPad Prism 9.0 software. One-way ANOVA was used to compare differences among groups. Pearson correlation coefficient was used to determine the correlation between antibody levels and neutralizing activity. Statistical significance was set at P < 0.05.
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