Immune response induced by spike protein from transmissible gastroenteritis coronavirus expressed in mouse mammary cells

Abstract

The present study is undertaken to investigate the immune response that was induced by the recombinant spike (S) protein from swine-transmissible gastroenteritis virus (TGEV) expressed in mouse mammary cells. A mammary-specific expression vector pEBS containing the full-length cDNA of S gene was constructed and expressed in the mouse mammary cells (EMT6). The recombinant S protein from culture supernatant of transgenic EMT6 was harvested and immunized BALB/c mice. The results demonstrated recombinant S protein was expressed at high levels in mammary cells by Western blotting and enzyme-linked immunosorbent assay (ELISA) detection. The antibody titer in BALB/c mice following immunization with recombinant S protein was detectable after the first immunization. Maximum titers of antibody (8.86+/-0.19 ng/ml of serum) were attained after the second immunization. In conclusion, the recombinant S protein expressed in mammary cells was able to elicit substantial immunological response against TGEV. This lays the basis for using mammary gland bioreactor generating edible vaccine.

Fig. 1

Schematic representation of recombinant plasmid pEBS. CMV Pro: CMV promoter; EGFP: enhance green fluorescence protein reporter gene; CSN2 Pro: bovine β-casein gene promoter; S gene: spike protein gene; PA: bovine β-casein gene 3′ polyadenylation signal.

Fig. 2

Flueofluence microscope image of positive cell clone (A, B, C and F = 60 μm; D, bar = 40 μm; E, bar = 20 μm). (A) Transfection group of pEBS. (B) Untransfection group. (C) Transfection group of pEGFP/C1. (D) Untransfection EMT-6 cells. (E and F) Positive clone of the transfection group.

Fig. 3

Identification of positive clones. (A) PCR analysis of positive clones. (B) S gene transcription in positive clones analyzed by RT-PCR. Samples were treated with RNase (lower) or not (upper). Lanes 1–3, transfection group of pEBS; lane 4, transfection group of pEGFP/C1; lane 5, untransfection group.

Fig. 4

ELISA analysis of recombinant S protein from culture supernatants. The figure shows the mean ± S.E. of the absorbance readings. 1–3, test group. Transfection group of pEGFP/C1 as negative control, purified TGEV virus as positive control. Test groups have higher absorbance than negative control (P < 0.01) and can be up to the level of TGEV. ΛA = absorbance 492 nm.

Fig. 5

Western blot analysis of recombinant S protein. 1: Purified TGEV virus; 2: untransfection group; 3: transfection group of pEGFP/C1; 4 and 5: transfection group of pEBS; M: protein molecular marker.

Fig. 6

Anti-TGEV S protein antibody titers induced after immunization with recombinant S protein. Sera from three groups of mice immunized orally with recombinant S protein or control group (pEGFP/C1) or PBS were tested by ELISA. Bars represent the mean ELISA titer ± S.E.M. in each group.

Fig. 7

Inhibition of viral plaque formation by sera from mice fed with recombinant S protein. Maximum reduction in number of plaque, expressed as a percentage of plaques obtained for the negative control samples, using sera collected from mice fed with recombinant S protein was 16 ± 0.8%. Results are mean values and standard errors of triplicates.