Journal: Journal of Virology

Article Title: Design of antibody structure-guided epitope vaccines in silico to induce potent immune responses against emerging viruses

doi: 10.1128/jvi.00689-25

Figure Lengend Snippet: Analysis of protein interactions of epitope peptides with scFv1. ( A ) Analysis of protein interaction between P30 peptide and scFv1. Plasmids pEGFP-P30 and pCMV-Flag-scFv1 were constructed and co-transfected into HEK293T cells. After 48 h, the proteins were collected and incubated with magnetic beads treated with mouse anti-GFP mAb. The samples were eluted with SDS-PAGE loading buffer and verified by Western blot. Plasmids pEGFP and pCMV empty vector were used as controls. ( B ) Analysis of protein interaction between S390F peptide and scFv1. ( C ) Analysis of protein interaction between G394K peptide and scFv1. ( D ) The self-activation verification of the bait plasmid PGBKT-scFv1 on SD-deficient medium. ( E ) Negative and positive controls in yeast two-hybrid assays. The pGADT-T plasmid was co-transformed with pGBKT-lam and pGBKT-53 plasmids in Y 2 Hgold competent cells as negative and positive controls, respectively. ( F ) The protein interactions between scFv1 and the P30, S390F, and G394K peptides were analyzed by yeast two-hybrid assay. After dilution, the bait plasmid pGBKT-scFv1 was co-transformed with the pGADT-P30, S390F, and G394K plasmids in Y 2 Hgold competent cells and cultured in SD-Leu-Trp and SD-Leu-Trp-His-Ade deficient medium for 48–96 h. ( G ) After a 10-fold dilution, the number of plaques on SD-LTHA deficient medium of competent cells co-transformed with pGADT-P30, S390F, and G394K with pGBKT-scFv1 plasmids was counted. * P < 0.05. ** P < 0.01. ( H ) After 100-fold dilution, the number of plaques on SD-LTHA deficient medium. * P < 0.05. ** P < 0.01. ( I ) After 1,000-fold dilution, the number of plaques on SD-LTHA deficient medium. * P < 0.05. ** P < 0.01. ( J ) The co-localization of P30, S390F, and G394K peptides with scFv1 was observed using laser confocal microscopy. To achieve this, pEGFP-P30, S390F, and G394K were co-transfected with pCMV-DsRed-scFv1 into HEK293T cells and cultured for 48 h. The fluorescence expression of the proteins was observed and analyzed under a 63× confocal microscope after DAPI staining with an anti-fluorescence quencher.

Article Snippet: In brief, TBS-washed protein A + G magnetic beads were incubated with GFP-tagged IgG2a mAb (1:3,000, Proteintech) for 1 h at room temperature.

Techniques: Construct, Transfection, Incubation, Magnetic Beads, SDS Page, Western Blot, Plasmid Preparation, Activation Assay, Transformation Assay, Y2H Assay, Cell Culture, Confocal Microscopy, Fluorescence, Expressing, Microscopy, Staining

Journal: mBio

Article Title: Ec CXCR4b influences RGNNV proliferation by interacting with the RGNNV capsid protein

doi: 10.1128/mbio.02045-25

Figure Lengend Snippet: Ec CXCR4b mediates RGNNV entry through the clathrin endocytosis pathway. ( A and B ) Effect of clathrin endocytosis pathway inhibitor on viral internalization in GS cells. GS cells were subjected to pretreatment with various doses of CPZ before the virus infection. Then the cells were collected to detect the expression level of RGNNV-CP through qRT-PCR and western blotting. ( C ) Effect of clathrin endocytosis pathway inhibitor on viral internalization in A549 cells. The Ec CXCR4b-transfected A549 cells were also subjected to the same treatment as described above, and the CP expression level was analyzed using a qRT-PCR assay. ( D ) Knockdown of endogenous clathrin expression via RNA interference. siRNA for clathrin or control siRNA (NC) was transfected into GS cells. After 24 h, qRT-PCR was employed to evaluate the knockdown efficiency of siRNAs targeting clathrin expression. ( E and F ) Effect of expression of clathrin on RGNNV replication in GS cells. Clathrin expression in GS cells was either downregulated or upregulated, and then the internalization assays were performed. The expression of RGNNV-CP was determined using qRT-PCR and western blotting. ( G and H ) The interaction of Ec CXCR4b and RGNNV-CP was investigated by Co-IP and intracellular immunofluorescence localization assays. GS cells were co-transfected with plasmid combinations indicated in the figures for 24 h Western blotting was used to examine the cell lysates after they had been immunoprecipitated using anti-GFP Abs ( G ). GS cells were transfected with pGFP- Ec CXCR4b/pcDNA4.0-Clathrin-6His plasmids for 24 h, then one group was incubated with virus (MOI = 10) for 4 h at 28°C, another group was a mock. The cells were then subjected to immunofluorescence staining using a specified antibody, and cell nuclei were stained with DAPI. The fluorescent signal was observed with a confocal laser scanning microscopy (bar = 20 µm) ( H ). Data represent means ± standard deviation. * P < 0.05, ** P < 0.01, and *** P < 0.001.

Article Snippet: Mouse anti-GFP mAb (66002-1-Ig) and mouse anti-His mAb (66005-1-Ig) were obtained from Proteintech (Wuhan, China); mouse anti-RGNNV-CP mAb was prepared and preserved in our laboratory ( ).

Techniques: Virus, Infection, Expressing, Quantitative RT-PCR, Western Blot, Transfection, Knockdown, Control, Co-Immunoprecipitation Assay, Immunofluorescence, Plasmid Preparation, Immunoprecipitation, Incubation, Staining, Confocal Laser Scanning Microscopy, Standard Deviation