| 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. E. coli strains caused edema disease and/or post-weaning diarrhoea adhere to the small intestine of pigs by means of fimbria F4 (K88) or F18. Shiga toxin 2 variant(Stx 2e) is also produced by these organisms. Immunoprophylaxis of porcine edema disease and post-weaning diarrhoea caused by strains of Escherichia coli expressing fimbriae F18 and/or F4 is still an unsolved problem. A potential recombinant vaccine strain, BL21(pFSFaeG) to prevent ED and PWD was constructed by Shi hui et al., which expressed two different subunits (FedA and FaeG)of both fimbriae, namely F18 and F4, and the subunit B of Shiga toxin 2 variant, using pGEX-6P-1 vector. Partly soluble 80kD GST-FedA-Stx2eB-FaeG fusion protein was expressed in the recombinant strain BL21(pFSFaeG) after induced by IPTG at 37℃, which was confirmed by SDS-PAGE and Western blotting analysis.By introducing asd gene from Sallmonella typhimurium into a prokaryotic expression plasmid vector of pGEX-6p-l vector to replace the ampicillin resistance gene, a new prokaryotic expression recombinant strain which contained no antibiotic resistance gene was constructed. Restriction endonuclease analysis was used to indentify the new recombinant plasmid. The results indicated that the plasmid wascorrectly constructed and the ampicillin resistance gene was koncked out, and the new recombinant strain propagated in vitro without the need of antibiotic pressure, and the stability of the plasmid and the expression of the fusion protein were confirmed very well.The 5-week-old mice were immunized with live recombinant strain BL21(pFSFaeG) orally and with the inactivated vaccine of recombinant strain BL21(pFSFaeG) by the peritoneal route. Sera and feces were simultaneously collected from the vaccined mice on the 7d, 14d post first immunization and on the 7d, 14d post second immunization. The result indicated that serum IgG specific to F4, F18 and Stx 2e antigens can be stimulated in mice immunized with inactivated vaccine, but not in all oral groups. The protection efficacy of mice immunized with inactivated recombinant strain against challenging of strain 107/86, C83907 and Stx 2e is much better than mice immunized with live bacteria orally.160 5-week-old mice (ICR) were divided into experimented group and control group separated. In experimented group, mouse were immunized with the inactivated vaccine of recombinant strain BL21 (pFSFaeG), inactivated bacteria of 107/86, C83907 and inactivated toxin of Stx 2e by the peritoneal route. Sera were collected from the vaccinated group and non-vaccinated group on the 7d, 14d post first immunization and on the 7d, 21d post second immunization. Titers of IgG from sera against F4, F18 and Stx 2e antigens were measured by indirect ELISA respectively. The result indicated that the recombinant strain BL21(pFSFaeG) seemed to stimulate serum IgG specific to F4, F18 and Stx 2e antigens, and the titers of IgG of the immunized mice reached its highest level on 21 day post second immunization, while that of IgA of the immunized mice was not detected. The protection efficacy of vaccinated mice with inactivated recombinant strain BL21(pFSFaeG) against challenging of strain 107/86, C83907 and Stx 2e toxin was 81.8%, 87.5% and 81.3% respectively.Stx 2e was purified from E.coli strain TB1 induced with polymyxin B by two-step ammonium sulphate precipitation, and only a single band of protein was visualized in SDS-PAGE, which seemed to be the A1 subunit of Stx 2e. BALB/c mice were immunized with the purufied Stx 2e mentioned above, then their spleen cells were fused with the SP2/0 cells, and there positive hybridomas were selected by indrectenzyme-linked immunosorbent assay(ELISA), which designated 2E11, 2H9, 4F3. The ELISA titers of supernatants collected from hybridoma 2E11, 2H9 and 4F3 were 1: 8192 , 1: 8... |
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