Bacterial strains, plasmids and growth conditions
The bacteria and plasmids are shown in Additional file 1. The bacterial cultivation conditions were as described previously [12, 14]. SPF BALB/c mice, aged 6 weeks, were purchased from Henan Experimental Animal Center (Zhengzhou, China).
Polymerase chain reaction of ltB gene
The ltB gene was amplified by PCR from the plasmid pMAL-c2x-mlt63 using Pyrobest DNA polymerase (TaKaRa, China). A pair of oligonucleotide primers was designed based on the published sequence (GenBank EF057802). The sequences of the primers were 5’-CAGTCGGCATGCGCTCCCCAGTCTATTAC-3’ (Sense) and 5’-CGCTCTAGACTAGTTTTCCATACTGATTG-3’ (Antisense) with the endonuclease sites SphI and XbaI shown in bold letters, respectively. The PCR profile included 30 cycles of 94 °C for 1 min, 55 °C for 30 s and 72 °C for 2 min.
Construction of recombinant L. lactis strains
The ltB gene was ligated with TA cloning vector pMD19-T and used to transform E. coli DH5α by heat shock. The ltB gene fragment was collected by digestion of pMD19-T-ltB using SphI and XbaI (TaKaRa, China), ligated with plasmid pNZ8149-SP (GenBank KY385376), and used for transformation of L. lactis NZ3900 by electrophoration [12]. The recombinants were obtained by Elliker medium selection, and identified by restriction digestion and gene sequencing as reported before [12]. The recombinant NZ3900 strain carrying ltB gene was referred to as L. lactis NZ3900/pNZ8149-SP-ltB.
LTB expression and western blotting assays
The expression of LTB in NZ3900/pNZ8149-SP-ltB was induced using 25 ng/ml nisin (Sigma, USA) as inducer under the conditions as previously described [12].
Samples of the culture supernatant were prepared from 50 ml of culture. The supernatant was obtained by centrifugation at 10,000 rpm for 20 min at 4 °C, and filtered through 0.22 μm filter. The proteins in the filtrate were precipitated by adding trichloroacetic acid (10%, v/v), incubating at 4 °C for 16 h and centrifugation at 10,000 rpm for 30 min at 4 °C. The pellet was resuspended in 8 ml acetone, centrifugated at 10,000 rpm for 20 min at 4 °C, then kept in fume hood at room temperature until dry. The protein sample was added 360 μl PBS, kept at 4 °C for 3 h, and centrifugated at 10,000 rpm 4 °C for 10 min. The supernatant was collected and used as samples of the culture supernatant. Samples of bacterial cell lysates were processed as described before [12].
SDS-PAGE and western blotting assays were performed using mouse anti-LTB antibody (Abcam, USA) as the primary antibody as previously reported [12].
Oral vaccination of mice
The mice were assigned at random into three groups of 10 each. For Lpp20 group and Lpp20 + LTB group, the mice were treated by gavage with cell suspensions at a dose of 300 μl of NZ3900/pNZ8149-SP-lpp20 (1 × 1011 CFU/ml) and a mixture of NZ3900/pNZ8149-SP-lpp20 (1 × 1011 CFU/ml) and NZ3900/pNZ8149-SP-ltB (1 × 1011 CFU/ml), respectively, on day 0, 7, 14, 21, 28 and 35. For PBS group, mice were given equal volumes of PBS instead of the cell suspensions.
Blood and intestinal feces sampling
Seven days after the last vaccination, blood and intestinal feces were collected for half number of the mice from all the groups. The blood samples were fetched from orbital sinus and kept at 4 °C for 16–20 h, and then the sera were separated and stored in aliquots at −20 °C. For sampling intestinal feces, the mice were sacrificed by spinal dislocation, 100 mg of feces was fetched from the intestine for each mouse, and then 1 ml of PBS containing proteinase inhabitor (Phenylmethanesulfonyl fluoride, 0.1 mM) was injected by the duodenum to wash the intestinal wall. The eluate was recovered, mixed with the feces and kept at 4 °C for overnight (14–16 h). The supernatant of the mixture was separated via centrifugation at 12,000 rpm for 10 min, and stored at −20 °C as ELISA samples.
ELISA detection of IgG and SIgA antibodies
The H. pylori-specific serum IgG and fecal SIgA antibodies were quantified by ELISA as described before [12]. Briefly, 96-well microplates (Beijing Solarbio, China) were coated with soluble H. pylori somatic proteins. The ELISA signals were developed using biotinylated goat anti-mouse IgG (Abcam, USA), goat anti-mouse SIgA (Abcam, USA) and p-nitrophenyl phosphate (PNPP) substrate (Beijing Solarbio, China). The absorbances of the wells at 450 nm (OD450) were measured using a Microplate Reader (Tecan Sunrise, CH), and designed as indicators of the specific sIgA and IgG levels.
Statistical analysis
The measurement data were presented as means ± standard deviation \( \left(\overline{x},\pm, s\right). \) The significance of the difference among the groups was tested using Kruskal-Wallis tests, while the pairwise comparisons of mean values were carried out using the Mann-Whitney U test with the aid of software SAS9.13. The difference was considered as significant at P <0.05.
