Helicobacter pylori HopE and HopV porins present scarce expression among clinical isolates

Figure 1 Cloning of H. pylori hopE and hopV genes. A: Polymerase chain reaction (PCR) amplification of hopE and hopV genes. Amplicons and plasmids were separated by 1% agarose gel electrophoresis. Lane Std: 1 kb DNA ladder standard; B: Detection of plasmids carrying the hopE gene. Lane 1: Strain AD494(DE3)pLysS with pET21d as control. Lanes 2, 3: Negative clones, lanes 4-7 plasmids carrying the hopE gene; C: Release of inserts carrying the hopE and hopV genes after NcoI/HindIII and NdeI/BamHI digestions respectively. Lane Std: 1 kb DNA ladder standard (Fermentas).

Figure 2 Expression of H. pylori hopE and hopV genes in Escherichia coli AD494(DE3)pLysS. Bacterial lysates were separated in 12%-15% PAGE-SDS gel, stained with Coomassie blue (A and B) or analyzed by Western blotting (C and D). Conditions for HopE expression are indicated below the figure (A, lanes 5 and 6; C, lane 2). Conditions for HopV expression are indicated under the figure (B, lanes 3 and 4; D, lane 2). Arrows indicate electrophoretic migration of these proteins. Lane Std: Prestained molecular weight marker in kDa (Fermentas).

Figure 3 Detection of hopE and hopV genes and their expression in H. pylori clinical isolates from different Chilean cities. Amplicons were separated in 1% agarose gels. A: PCR detection of the hopE gene; B: PCR detection of the hopV gene. Arrows indicate migration of the respective gene fragments. Lane Std: 1 kb DNA ladder standard (Fermentas); Lane Std2: Lambda DNA/HindIII marker (Fermentas); C: Expression of HopV and HopE porins in H. pylori Chilean strains separated by 12% SDS-PAGE gels and detected by Western blots with respective polyclonal antibodies. Clinical isolates are indicated under the respective lanes. Std: Prestained molecular weight marker (Fermentas). The CHCTX-1 strain was included as a positive control.

Figure 4 Frequency of recognition of 4 H. pylori antigens by human sera. Bars represent number of immunoreactive human sera from a panel of 69 H. pylori-infected patients showing IgG- and IgA-type immunoreactivity tested by Western blotting assays on E. coli lysates expressing separately HopE, HopV, CagA and VacA cloned antigens. A: Frequency of IgA- and IgG-type immune responses (as number of sera) which reacted with lysates containing one of these antigens; B: IgA response data taken from panel A, with patients separated by age into 2 groups: under 18 years old and adults; C: IgG response data taken from panel A, separated by age as above. As negative controls, sera from 8 non-infected patients did not display any immune response when tested with these antigens (not shown). Fisher’s test was used for statistical analysis, and significance lower than 0.05 is indicated.