Evaluation on the efficacy and immunogenicity of recombinant DNA plasmids expressing S gene from porcine epidemic diarrhea virus and VP7 gene from porcine rotavirus

Abstract

Porcine rotavirus (PoRV) and porcine epidemic diarrhea virus (PEDV) usually co-infect pigs in modern large-scale piggery, which both can cause severe diarrhea in newborn piglets and lead to significant economic losses to the pig industry. The VP7 protein is the main coat protein of PoRV, and the S protein is the main structural protein of PEDV, which are capable of inducing neutralizing antibodies in vivo. In this study, a DNA vaccine pPI-2.EGFP.VP7.S co-expressing VP7 protein of PoRV and S protein of PEDV was constructed. Six 8-week-old mice were immunized with the recombinant plasmid pPI-2.EGFP.VP7.S. The high humoral immune responses (virus specific antibody) and cellular immune responses (IFN-γ, IL-4, and spleen lymphocyte proliferation) were evaluated. The immune effect through intramuscular injection increased with plasmid dose when compared with subcutaneous injection. The immune-enhancing effect of IFN-α adjuvant was excellent compared with pig spleen transfer factor and IL-12 adjuvant. These results demonstrated that pPI-2.EGFP.VP7.S possess the immunological functions of the VP7 proteins of PoRV and S proteins of PEDV, indicating that pPI-2.EGFP.VP7.S is a candidate vaccine for porcine rotaviral infection (PoR) and porcine epidemic diarrhea (PED).

Keywords: Co-expression; DNA vaccine; Immune effect; PEDV; PoRV.

Fig. 1

A Identification of the recombination plasmid pPI-2.EGFP.VP7.S using PCR. Lane M: 2000 bp control size DNA markers lane 1: S gene fragment of PEDV amplified through PCR from pPI-2.EGFP.VP7.S. lanes 2 and 3: Negative control. Lane 4: VP7 gene fragment of PoRV amplified through PCR from pPI-2.EGFP.VP7.S. B Identification of the recombination plasmid pPI-2.EGFP.VP7.S by restriction enzyme digestion. Lane 1: pPI-2.EGFP.VP7.S was digested with EcoR I and BamH I released an insert size 1967 bp and pPI-2.EGFP vector(7611 bp). Lane 2: pPI-2.EGFP.VP7.S was digested with Kpn I and BamH I released an insert size 989 bp and pPI-2.EGFP.VP7 (8600 bp). Lane 3: PPI-2.EGFP.VP7.S undigested vector. Lane M: 10000 bp control size DNA markers

Fig. 2

A: SDS-PAGE detection the expression of pPI-2.EGFP.VP7.S in BHK-21 cells. Lane 1: BHK-21 cells were transfected with pPI-2.EGFP.VP7.S. Lane 2: Negative cells. Lane M: protein marker. B: Western Blot detection the expression of pPI-2.EGFP.VP7.S in BHK-21 cells. Lane 1: Primary antibodies were antiserum of mouse to PEDV. Lane 2: Primary antibodies were antiserum of mouse to PoRV

Fig. 3

Humoral immune responses of the mice immunized with 100 μg pPI-2.EGFP.VP7.S intramuscular injection (group A), 100 μg pPI-2.EGFP.VP7.S subcutaneous injection (group D). In addition, the empty vector pPI-2.EGFP (group H), PBS (group K), PoRV SC201201 (group I), and PEDV CV777 (group J) were used as controls. Anti-PoRV serum antibodies (a) and anti-PEDV serum antibodies (b) were detected by ELISA at different time points following the injection of the plasmid DNAs. Different capitals at the same time represent the difference was extremely (P < 0.01), different small letters represent the difference was stastically (P < 0.05)

Fig. 4

Humoral immune responses of the mice immunized with 200 μg (group B), 100 μg (group A), 50 μg (group C) pPI-2.EGFP.VP7.S, respectively. In addition, the empty vector pPI-2.EGFP (group H), PBS (group K), PoRV SC201201 (group I), and PEDV CV777(group J) were used as controls. Anti- PoRV serum antibodies (a) and anti-PEDV serum antibodies (b) were detected by ELISA at different time points following the injection of the plasmid DNAs. Different capitals at the same time represent the difference was extremely (P < 0.01), different small letters represent the difference was stastically (P < 0.05)

Fig. 5

Humoral immune responses of the mice immunized with 2000 U pig IFN-α (group E), pig IL-12 (group F) and 300 ul pig spleen transfer factor (group G) at 24 h and 48 h after first and secondary immunity. In addition, 100 μg pPI-2.EGFP.VP7.S intramuscular injection (group A), PBS (group K), PoRV SC201201 (group I), and PEDV CV777 (group J) were used as controls Anti- PoRV serum antibodies (a) and anti- PEDV serum antibodies (b) were detected by ELISA at different time points following the injection of the plasmid DNAs. Different capitals at the same time represent the difference was extremely (P < 0.01), different small letters represent the difference was stastically (P < 0.05)

Fig. 6

The mice were immunized with different doses, approaches, and adjuvant. In addition, the empty vector pPI-2.EGFP, PBS, PoRV, and PEDV were used as controls. The proliferation of mouse spleen lymphocytes.was analyzed by MTT assays with ConA. The y-axis represents the lymphocyte proliferation levels (OD490). Different capitals at the same time represent the difference was extremely (P < 0.01), different small letters represent the difference was stastically (P < 0.05)