Fig 1.
Construction of pLATE31-vapD-6xHis.
The vapD gene lacking a stop codon was cloned into a pLATE31 vector. This expression vector presents a six histidine coding sequence (6xHis), which allows the expression of the protein with a C-terminal 6xHis-tag. Additionally, the vector presents the following characteristics: a β-lactamase gene (bla) conferring resistance to ampicillin (APR); an origin of replication (rep [pMB1]); the rop protein gene, which regulates the number of copies of the plasmid; the lac repressor (lacl), which ensures a strict control of the basal expression of the T7 RNA polymerase promoter (PT7); the T rrnBT1-2 transcription terminator, which prevents basal gene expression from vector derived promoter-like elements; the lacO operator, which ensures a strict control of gene expression; a ribosome binding site (RBS); Ptet, a promoter reduces basal expression from the PT7; the T7 terminator (TT7), which terminates transcription from the PT7. The vector was created in BioRender (https://BioRender.com).
Fig 2.
Expression of rVapD in E. coli Rosetta (DE3).
Aliquots of 20 µl of culture were collected at 0, 1, 2, 3, and 4 h after the addition of 0.1mM IPTG to verify the correct expression of rVapD by SDS-PAGE. MW, molecular weight.
Fig 3.
Purification and identification of rVapD by IMAC.
A. Recuperation of rVapD. Aliquots of 20 µl from each elution were separated by SDS-PAGE. Lane 1: flow-through of the E. coli Rosetta (DE3) lysate after the His-tag column; lanes: 2-3, washes with buffer containing 50 mM imidazole; lanes: 4-8, rVapD eluted from the His-tag column with buffer containing 325 mM imidazole. B. rVapD was concentrated after IMAC and confirmed by Western blotting anti-His using 1 µg of rVapD. MW, molecular weight.
Fig 4.
Use of non-denaturing detergents in rVapD purification.
A. Recovery of rVapD. Lanes 1 and 2: soluble (20 µg) and insoluble (2 µl) fractions, respectively, from E. coli Rosetta (DE3) lysate overexpressing rVapD with lysozyme method. Lanes 3 and 4: soluble (20 µg) and insoluble (2 µl) fractions from E. coli Rosetta (DE3) lysate overexpressing rVapD with lysozyme and non-denaturing method, respectively. B. Purification of rVapD by IMAC after non-denaturing method. Aliquots of 20 µl from each elution were separated by SDS-PAGE. Lane 1: flow-through of the E. coli Rosetta (DE3) lysate after the His-tag column; lanes: 2-3, washes with buffer containing 50 mM imidazole; lanes: 4-8, rVapD eluted from the His-tag column with buffer containing 325 mM imidazole. MW, molecular weight.
Fig 5.
Production and testing of polyclonal antibodies against rVapD.
A. Representation of the immunization schedule. B. Absorbance values of the indirect ELISA with serum tested at 30, 45 and 60 days (gray arrows in A), rVapD was used at a final concentration of 100 ng. C. Dot blotting test of the final bleeding (red arrow in A) using three dilutions of the serum (1:500, 1:1,000, and 1:2,000) and a serially two-fold diluted concentration of rVapD (1 to 0.03 µg). D. Western blotting of the serum at a dilution of 1:2,000 against a serially two-fold diluted concentration of rVapD (1 to 0.12 µg). Fig 5A was created in BioRender (https://BioRender.com).
Fig 6.
Detection of VapD from H. pylori in vivo by immunofluorescence assay.
H pylori was observed in AGC cells at 48 h post-inoculation. The expression of VapD coincided with the presence of the bacterium. The arrows point to vesicles where H. pylori is internalized.
Fig 7.
Lane 1: mouse serum (5 µl) + bacterial lysate (20 µl); lane 2: mouse pre-immune serum (5 µl) + bacterial lysate (20 µl); lane 3: lysate of Rosetta (DE3) cells overexpressing rVapD (20 µl). MW, molecular weight.