The construction of recombinant Lactobacillus casei vaccine of PEDV and its immune responses in mice

Abstract

Background: Porcine epidemic diarrhea (PED) is a contagious intestinal disease caused by porcine epidemic diarrhea virus (PEDV) characterized by vomiting, diarrhea, anorexia, and dehydration, which have caused huge economic losses around the world. At present, vaccine immunity is still the most effective method to control the spread of PED. In this study, we have constructed a novel recombinant L. casei-OMP16-PEDVS strain expressing PEDVS protein of PEDV and OMP16 protein of Brucella abortus strain. To know the immunogenicity of the recombinant L. casei-OMP16-PEDVS candidate vaccine, it was compared with BL21-OMP16-PEDVS-F, BL21-OMP16-PEDVS, and BL21-PEDVS recombinant protein.

Results: The results showed that we could detect higher levels of IgG, neutralizing antibody, IL-4, IL-10, and INF-γ in serum and IgA in feces of L. casei-OMP16-PEDVS immunized mice, which indicated that L. casei-OMP16-PEDVS candidate vaccine could induce higher levels of humoral immunity, cellular immunity, and mucosal immunity.

Conclusion: Therefore, L. casei-OMP16-PEDVS is a promising candidate vaccine for prophylaxis of PEDV infection.

Keywords: Immune responses; Lactobacillus casei vaccine; OMP16; PEDV; PEDV S protein.

Fig. 1

The enzyme digestion results of recombinant plasmids. A: The enzyme digestion results of the pVE5523-OMP16-PEDVS plasmid. M: D8000 DNA Ladder Marker; 1: pVE5523-OMP16-PEDVS plasmid. B: The enzyme digestion results of pet28a-OMP16-PEDVS and pET28a-PEDVS plasmid. M: D8000 DNA Ladder Marker; 1: pet28a-OMP16-PEDVS plasmid; 2: pET28a-PEDVS plasmid

Fig. 2

The verification of recombinant expression proteins. M: protein marker; 1: the secretory protein form recombinant L. casei-pVE5523-OMP16-PEDVS strain; 2: the purified protein from BL21-pET28a-OMP16-PEDVS strain; 3: the purified protein from BL21-pET28a-PEDVS strain

Fig. 3

The IgG antibody levels of candidate vaccines in the serum of immunized mice. Serum was collected on days 0, 14, and 28 days before or after immunization and examined via commercial ELISA kits and measured at an absorbance of 450 nm. Bars represent the mean ± standard deviation of three independent experiments. * p < 0.05, ** p < 0.01, and **** p < 0.0001 represent increasing degrees of significant differences, respectively, and ns means no significant difference

Fig. 4

The IgA antibody levels of candidate vaccines in feces of immunized mice. Feces were collected on day 0, 14, and 28 days before or after immunization and examined via commercial ELISA kits and measured at an absorbance of 450 nm. Bars represent the mean ± standard deviation of three independent experiments. * p < 0.05, ** p < 0.01, and **** p < 0.0001 represent increasing degrees of significant differences, respectively, and ns means no significant difference

Fig. 5

The neutralizing antibody levels of candidate vaccines in the serum of immunized mice. Serum was collected on days 0, 14, and 28 days before or after immunization and examined via neutralization test. Bars represent the mean ± standard deviation of three independent experiments. * p < 0.05, ** p < 0.01, and **** p < 0.0001 represent increasing degrees of significant differences, respectively, and ns means no significant difference

Fig. 6

Detection of cytokine levels from the serum of immunized mice. Serum was collected on days 0, 14, and 28 days before or after immunization and examined via commercial ELISA kits. The absorbance value was measured at an absorbance of 450 nm for IL-4 (a), IL-10 (b), and IFN-γ (c), respectively. Bars represent the mean ± standard deviation of three independent experiments. * p < 0.05, ** p < 0.01, and **** p < 0.0001 represent increasing degrees of significant differences, respectively