Construction Of Recombinant Strain Expressing The Major Protective Antigens Of Shiga-like Toxigenic Escherichia Coli Caused PWD And ED

Porcine edema disease (ED) and post-weaning diarrhoea (PWD) caused by Shiga-like toxigenic Escherichia coli are the most common causes of mortality of recently weaned pigs. No effective methods for control of these two diseases are available. The organism has two types of virulent factors, fimbrial adhesins and Shiga-like toxin/enterotoxin. Fimbria, the primary virulent factor of the organism, is responsible for its adhesion to the enterocye receptor of piglets. The Shiga-like toxin/enterotoxin produced by E. coli is absorbed through the intestinal wall, which results in the damage of the target tissue. E. coli causing edema disease and/or post-weaning diarrhoea attaches to the small intestine by means of fimbria F4 (K88) or F18. Shiga-like toxin 2e(Stx 2e) is also produced by above organism. It has been demonstrated that oral administration of purified F4 can induce protective mucosal immunity in pigs. However, effective vaccines against ED and PWD are not available presently. In this study we constructed a recombinant subunit vaccine to prevent ED and PWD, which expressed two different subunits of both fimbriae, namely F18 and F4, and the subunit B of Shiga-like toxin 2e, using pGEX-6P-l vector.The gene faeG encoding the major fimbrial subunit of F4 was amplified by polymerase chain reaction (PCR) from the plasmid of strain C83907. The F18 major fimbrial subunit gene defined as fed A, which encodes of the major subunit protein FedA, was amplified by PCR from the plasmid of strain 107/86. The gene of stx 2eB was amplified from the plasmid of E. coli TB1. These fragments mentiond above were cloned into pGEM-T easy vector and then were subcloned into the same expression plasmid pGEX-6P-l. PCR, restriction endonuclease analysis and DNA sequencingwere used to identify the recombinant plasmid containing gst-fedA-stx2eB-faeG fusion gene. The results indicated that the plasmid was correctly constructed. Then the plasmid was transformed into E. coll BL21. Partly soluble 80kD GST-FedA-Stx2eB-FaeG fusion protein was expressed in recombinant strain BL21(pFSFaeG) after induced by IPTG at 37 C, which was confirmed by SDS-PAGE and Western blotting analysis.Twenty-two 5-week-old piglets were divided into experimented group and control group separated. In experimented group, piglets were vaccinated with the recombinant strain BL21(pFSFaeG) by the intramuscular route or oral route. The E. coli strains which resistant to ampicillin isolated from the oral immunized pigs using rectal swabs on the 3d, 7d, 10d, 14d post-inoculation period were identified by the recombinant plasmid analysis. The result showed that the recombinant strain BL21(pFSFaeG) was able to live in the gut of experiment-group's piglets stably during the observation period. Sera and feces were simultaneously collected from the vaccined pigs at 1,2, 4and 6 weeks after the last vaccination and liters of IgG and IgA from the sera and feces, respectively, against F4/F18 antigens were measured by indirect ELISA. The result indicated that the recombinant strain BL21(pFSFaeG) seemed to stimulate serum IgG specific to F4 antigen while not do the mucosal immune response to F4 or F18 antigens.