Modification And Expression Of A Subunit Of Shiga-Like Toxin Type ⅡVariant And Their Potential For Use In Prevation

Edema disease (ED) of weaning piglets is caused mainly by pathogenic Escherichia coil which produce Shiga-like toxin type II variant (SLII-lle). SLT-lle is composed of one A subunit and five B subunits The A subunit possesses RNA N-giycosidase activity, which specifically removes an acienine in the 28S subunit of eukaryotic rRNA, leading to inactivation of the cellular protein synthesis and disorganization of metabolic functions of the cell. Presently, effective vaccines against ED are not available. To construct effective recombinant vaccines against ED, three genetically modified SLT-lIe A subunit was generated by deleting its signal peptide, introducing double point mutations at residues 167(Glu-Gln) and 170 (Arg-Lys), and deleting l2bp corresponding to position 806-817 by PCR. The PCR products were cloned into prokaryotic expression vector pGEX-6P-1 and resulting recombinants were called pGEX-A~, pGEX-A,. and pGEX-A~ respectively. The three recombinant plasmids were transformed into E-coli BL2I and three GST-fusion proteins (GST-TJeA~,, GST-1IeA~, GST-lIeA.,) with respected sizes were expressed by Western blotting using monoclonal antibody specific for SLT-lle A. ICR mice were immunized twice with the sonicated E.coli expressing the three mutated A subunits of SLIt-lie and the immunized sera were tested with ELISA titers of 2抯, 2?and 2~ respectively and then challenged with purified wild type of the SLT-lle respectiveiy. 800/oof the mice immunized with GST-Iie A?and 100% of the mice immunize with GST-lIeAmu or GST:IleA.., were survived the challenge. And the neutralization titers of immunized scm against Shiga-like toxin II variant were 2?2?2~ respectively. The supernatant of sonicate E.coli transformed by recombinant plasmid respectively was filtered at 0.4Sum to devoid bacteria, then diluted to inoculate Vero cells, resulted in cell death, significant growth suppression, and no apperance altercation each by OST-11eA,~,, GST-lleAmu and GST-IIeA~. The above reserch is the first report on SLT-lIeA of ED, and will lay foundation to the prevation of ED by gene engineer vaccines.