Construction of recombinant Shiga-like toxin-IIv (SLT-IIv) and its use in monitoring the SLT-IIv antibody status of pigs.
We constructed and purified recombinant B-subunits of the SLT-IIv as well as tested their usefulness in an immunoblot assay. The slt-IIvB gene amplified by PCR was ligated into the fusion vector pGEX-2T, and expressed in E. coli K 12 laboratory strains. Deletion of the signal sequence was necessary for optimal expression. High quantities of the fusion protein could be purified by affinity chromatography and subsequently used as antigen for immunoblot analysis with serum samples from diseased pigs and healthy controls. IgG antibodies against SLT-IIv were detected in the sera of 11 of 52 (21.15%) healthy pigs. By contrast, only in 1 of 28 (3.57%) serum samples of pigs with edema disease caused by SLT-IIv-producing E. coli we could demonstrate SLT-IIv-specific antibodies. During an outbreak of edema disease, sera from 10 pigs were taken at 4, 20, and 40 days after disease onset to investigate the immune response elicited by SLT-IIv. Immunoblot analysis with the recombinant SLT-IIv fusion protein revealed that the number of IgG-positive serum samples increased within this period of 40 days from one on day 4, to seven on day 20, to ten on day 40; the number of IgM-positive samples also increased from one after 4 days to eight after 20 days. Forty days after disease onset, IgM reactivity was no longer detectable. Since all animals seroconverted in the follow-up sera, the antigenicity of SLT-IIv during infection of pigs seems to differ from that of SLT-II in human hemolytic uremic syndrome where only a minority of patients are known to mount an immune response. The recombinant SLT-IIvB described here may be a possible candidate for vaccination trials.
Franke
S
S
Institut für Hygiene und Mikrobiologie, Universität Würzburg, Germany.
Gunzer
F
F
Wieler
L H
LH
Baljer
G
G
Karch
H
H
D000818:Animals; D000907:Antibodies, Bacterial / Q000097:blood*; D001427:Bacterial Toxins / Q000096:biosynthesis* / Q000276:immunology*; D001483:Base Sequence; D004487:Edema / Q000276:immunology / Q000662:veterinary*; D004926:Escherichia coli; D007074:Immunoglobulin G / Q000097:blood; D007075:Immunoglobulin M / Q000097:blood; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000662:veterinary; D011994:Recombinant Proteins / Q000096:biosynthesis*; D022641:Shiga Toxin 2; D013552:Swine; D013553:Swine Diseases / Q000276:immunology*
D000907:Antibodies, Bacterial; D001427:Bacterial Toxins; D007074:Immunoglobulin G; D007075:Immunoglobulin M; D011994:Recombinant Proteins; D022641:Shiga Toxin 2