signal peptide  (Sangon Biotech)

Journal: Cells

Article Title: A Nanobody-Based Toolbox to Probe ApoE4 in the Secretory Pathway and Cytosol

doi: 10.3390/cells15050479

Figure Lengend Snippet: ER-targeted apoE nanobodies 9-74, 19-38, 17-69 and 18-91 bind apoE4 intracellularly and promote its intracellular retention in HEK293T cells. ( A ) Schematic representation of the ER-targeted nanobody construct containing an N-terminal mouse IgH secretion signal peptide (SEC2), a C-terminal HA-tag, and a KDEL ER retention sequence. ( B ) ApoE4 expression construct containing an N-terminal 18-amino-acid signal peptide (SEC1). ( C ) Schematic depiction of cells expressing apoE4 and an ER-targeted apoE nanobody. The KDEL-equipped nanobody is directed to the ER, allowing its interaction with apoE4 as it passes through the secretory pathway. ( D ) Microscopy images showing colocalization (yellow) of apoE4 (green) and ER-targeted nanobodies (Nb-KDEL; red) in HEK293T cells. Scale bar: 10 µm. ( E ) Scatter dot plot of the averaged Pearson’s correlation coefficient (PCC averaged ) values quantifying colocalization between apoE4 and each ER-targeted nanobody. Each dot represents the average PCC value of one independent experiment (~15 cells per experiment). Black horizontal lines and error bars indicate the mean ± standard deviation (SD; n = 3). All nanobodies displayed PCC averaged values significantly greater than 0.5 (red dotted line) and approaching 1, indicative of strong colocalization (Fisher Z-transformation followed by a one-sample t -test against a reference value of 0.5). * p < 0.05; ** p < 0.01; *** p < 0.001. ( F ) Co-IP assays showing interaction between each of the ER-targeted apoE nanobodies (Nb-KDEL) and apoE4 in cell extracts of co-transfected HEK293T cells (lanes 6). Co-IP was performed using anti-HA agarose beads and analyzed by Western blot. Two negative controls were included: NC1, a co-IP using the extract of cells transfected with apoE4 alone (lanes 4), and NC2, a co-IP using the extract of cells co-transfected with apoE4 and an ER-targeted GFP nanobody (lanes 5). For each condition, the corresponding input (cell extract) is shown (lanes 1–3). Uncropped blots are shown in . ( G ) Western blot analysis showing increased apoE4 levels in cell extracts (left) and reduced apoE4 levels in conditioned medium (right) from HEK293T cells co-transfected with apoE4 and each of the ER-targeted apoE nanobodies, compared to cells co-transfected with apoE4 and an ER-targeted GFP nanobody control. Actin was used as a loading control. Uncropped versions of the shown blots, together with replicate blots (n = 3), are provided in . ( H ) Quantification of intracellular apoE4 levels (cell extract Western blots). ApoE4 levels were normalized to actin and expressed relative to the GFP nanobody control within each blot/replicate. Individual data points are shown as dots (n = 3); bars represent mean ± SD. Raw data are provided in . ( I ) Quantification of secreted apoE4 levels (conditioned medium Western blots). ApoE4 levels were expressed relative to the GFP nanobody control within each blot/replicate. Individual data points are shown as dots (n = 3); bars represent mean ± SD. Raw data are provided in .

Article Snippet: The pCMV4-ApoE4 plasmid, encoding apoE4 with an N-terminal 18-amino-acid secretion signal peptide (SEC1), was obtained from Addgene (Watertown, MA, USA, cat. no. 87087).

Techniques: Construct, Sequencing, Expressing, Microscopy, Standard Deviation, Transformation Assay, Co-Immunoprecipitation Assay, Transfection, Western Blot, Control

Journal: Viruses

Article Title: mRNA Vaccine Against Japanese Encephalitis Virus Genotype IV Protects Against Lethal Infection

doi: 10.3390/v18020171

Figure Lengend Snippet: mRNA vaccine construction. ( a ) Schematics illustrating design of the JEV NSW2022 GIV vaccine mRNA constructs. Two mRNA vaccine constructs were generated, one with the Native signal peptide sequence, and one with a Shorter peptide sequence, which is based on a Moderna ZIKV mRNA vaccine. UTR HBB—untranslated regions of human beta globin. (For full sequence details see ). ( b ) Final purified mRNA species for the two vaccines analyzed by capillary electrophoresis. Sizes by nucleotide (nt) length illustrated by electropherograms and simulated gel images. ( c ) HEK293 cells were transfected with the vaccine mRNA species and after 24 h cells and supernatants were harvested. Cells and proteins precipitated from supernatants were loaded onto 3 parallel SDS-PAGE gels (35 µg protein per lane). Left—Western blotting using anti-envelope (E) monoclonal antibody, 4G2; blue dotted line—lining up markers (KDa) with E. Middle—SDS-PAGE gels stained with Coomassie blue (red marker is 70 KDa). Right—Western blotting using anti-GAPDH (housekeeping protein, expected molecular weight ≈ 37 KDa). Text labeling of the lanes is color coded to facilitate comparisons. ( d ) Final LNP vaccines with encapsulated mRNA or empty (Control). The mean diameter (Z-average) and polydispersity index (PdI) are provided for each vaccine.

Article Snippet: Herein, we evaluated two signal peptide sequences, the full-length 23 amino acid (a.a.) Native signal peptide sequence from JEV NSW2022 , and a Shorter 16 a.a. sequence from JEV NSW2022, with the latter based on a JEV signal peptide sequence used for a ZIKV mRNA vaccine (Moderna, mRNA-1893) [ , ] ( a and ).

Techniques: Construct, Generated, Sequencing, Purification, Vaccines, Electrophoresis, Transfection, SDS Page, Western Blot, Staining, Marker, Molecular Weight, Labeling, Control

Journal: Viruses

Article Title: mRNA Vaccine Against Japanese Encephalitis Virus Genotype IV Protects Against Lethal Infection

doi: 10.3390/v18020171

Figure Lengend Snippet: Mouse experiments. ( a ) Time line of vaccination, challenge, weight change, disease monitoring, and sample collection. ( b ) End point ELISA titers after the first vaccination (serum collected week 3) and after the second vaccination (serum collected week 8), with the indicated mRNA doses. Limit of detection 1 in 30 dilution; ND—not detected. Statistics by Mann–Whitney U tests. ( c ) Reciprocal 50% neutralization titers for the same serum samples as those described in ( b ), plus Imojev included as a positive control. Neutralization of JEV NSW2022 (left), with neutralization of JEV Nakayama (GIII isolate) (right) also shown for serum samples from mice receiving two doses of 5 µg. Limit of detection 1 in 20 (left) or 1 in 10 (right) serum dilution; ND—not detected. Statistics by Kolmogorov–Smirnov exact tests. ( d ) Percent body-weight change post-challenge for the mice described in c. Lines represent mean body weigh change, except for three mice which were plotted individually ( n = 1). These were the one out of five mice that survived from the 1 µg Shorter group (blue line) and the 1 µg Native group (red line); and the one mouse that did not survive in the 5 µg Shorter group (purple line). † indicates mice reached the clinically defined end point for euthanasia. ( e ) Kaplan–Meier plot of survival for the mice shown in d. Statistics by log rank statistic; compared with LNP control p < 0.023 for the 1 µg groups, and p = 0.005 for the 5 µg groups and Imojev. ( f ) Mean viremia post-challenge for the mice shown in ( e ) (legend as in ( e )). For the LNP Control group on day 3, n = 2. Dotted line—limit of detection for individual mice ≈ 2 log 10 CCID 50 /mL; ND—not detected. Statistics by Kolmogorov–Smirnov exact tests for day 2 relative to LNP control; for all groups p = 0.008.

Article Snippet: Herein, we evaluated two signal peptide sequences, the full-length 23 amino acid (a.a.) Native signal peptide sequence from JEV NSW2022 , and a Shorter 16 a.a. sequence from JEV NSW2022, with the latter based on a JEV signal peptide sequence used for a ZIKV mRNA vaccine (Moderna, mRNA-1893) [ , ] ( a and ).

Techniques: Enzyme-linked Immunosorbent Assay, MANN-WHITNEY, Neutralization, Positive Control, Control