lactobacillus paracasei bl23  (ATCC)

Journal: Pharmaceutics

Article Title: Lactobacilli as a Vector for Delivery of Nanobodies against Norovirus Infection.

doi: 10.3390/pharmaceutics15010063

Figure Lengend Snippet: Figure 1. Production of VHH 1E4-displaying Lactobacillus. (a) Diagram of the expression cassette (pAF900-1E4) for cell-surface display of 1E4. VHH on L. paracasei BL23. apf Pro, the promoter of apf gene from L. crispatus M247; apf RBS, ribosomal binding site of apf gene; apf SP, the signal peptide of apf gene; prtP anchor, the 243 C-terminal amino acids of prtP from L. paracasei BL23; apf C-ter, C-terminal part of apf gene (not translated). The transcriptional terminator is indicated as the stem-and-loop structure. (b) Production of VHH 1E4 in cell extract (CE) and culture supernatant (SC) of cell wall-anchored recombinant L. paracasei BL23 strains tested by Western blotting using monoclonal mouse anti-E-tag antibody and horseradish peroxidase-labeled goat anti-mouse antibody as primary and secondary antibodies, respectively. Lactobacillus paracasei BL23 cells transformed with the empty vector pIAV7 were used as a negative control. The red arrow indicates the band corresponding to VHH 1E4 fused to the cell wall anchor region of PrtP. (c) Cell surface display of VHH 1E4 in recombinant L. paracasei BL23 strains according to ELISA assay using bacterial suspensions, a monoclonal mouse anti-E-tag antibody as the primary antibody, and alkaline phosphatase-conjugated rabbit anti-mouse antibody as the secondary antibody. Non-transformed L. paracasei BL23 cells and the strain transformed by the empty vector pIAV7 were used as negative controls. Signals were detected by reading the absorbance at 405 nm. The experiment was performed twice, and results are expressed as mean ± 1 standard deviation (SD). (d) Plasmid persistence of the recombinant L. paracasei BL23 strains expressing VHH 1E4 (BL23-1E4), measured according to colony count assays. The experiment was performed twice, and results are expressed as mean ± 1 SD. hpi, hours post-inoculation.

Article Snippet: Lactobacillus paracasei BL23 (previously known as L. casei or L. zeae ATCC 393 pLZ15−) cells were grown anaerobically at 37 ◦C on MRS agar plates (Difco, Becton Dickinson, Sparks, MD, USA) or in MRS broth without shaking.

Techniques: Expressing, Binding Assay, Recombinant, Western Blot, Labeling, Transformation Assay, Plasmid Preparation, Negative Control, Enzyme-linked Immunosorbent Assay, Standard Deviation

Journal: Pharmaceutics

Article Title: Lactobacilli as a Vector for Delivery of Nanobodies against Norovirus Infection.

doi: 10.3390/pharmaceutics15010063

Figure Lengend Snippet: Figure 3. The expression of VHH 1E4 on the surface of lactobacilli collected from the feces of germ-free mice before and after oral administration with strain BL23-1E4. Flow cytometry was used to confirm the display of VHH on the surface of Lactobacillus strains. (a) Lactobacilli in the feces of germ-free mice before and after oral administration of L. paracasei BL23-pIAV7 or BL23-1E4 were stained with thiazole orange (TO), whereas 1E4 was detected via E-tag by using rabbit anti-E-tag IgG followed by brilliant violet 421-labelled goat anti-rabbit IgG. The purple box indicates the cell population that is double positive for TO and E-tag-associated 1E4. (b) The proportion (%) of E-tag-positive cells among TO-positive cells (i.e., live bacteria) is shown at various times (h) after oral administration of germ-free BALB/c mice with L. paracasei BL23-pIAV7 or BL23-1E4. The experiment was performed three times, and the results are shown as mean ± 1 SD.

Article Snippet: Lactobacillus paracasei BL23 (previously known as L. casei or L. zeae ATCC 393 pLZ15−) cells were grown anaerobically at 37 ◦C on MRS agar plates (Difco, Becton Dickinson, Sparks, MD, USA) or in MRS broth without shaking.

Techniques: Expressing, Flow Cytometry, Staining, Bacteria