Preparation Of Monoclonal Antibody Against A Subunit Of Shiga-Like Toxin Type Ⅱ Variant (SLT-ⅡeA) And Coexpression Of A Subunit Of Fimbriae F18ab And SLT-ⅡeA

Edema Disease (ED) of weaning piglets is caused mainly by Verotoxigenic Escherichia coli (VTEC) which lead to edema in various tissues including the central nervous system. The name 'Edema Disease' was first used because edema of the stomach submucosa and the mesocolon were often a prominent feature of the disease. Fimbriae F18ab and variant of the SLT-IIe toxin were two pathogenic factors of VTEC,The E. coli adheres and ecesises in the mesocolon which make the SLT-IIe toxin enter the economy. SLT-IIe is composed of one A subunit and five B subunits.The A subunit possesses RNA N-glycosidase activity, which specifically removes an adenine 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, so researches of coexpression of them has importance to the prevention of ED.In order to identify the production of co-expression,it is necessary to have the monoclonal antibody . The toxin SLT-IIe was difficult to purify, so we expressed fusion protein GST-SLT-IIeA in recombinant E.coli pSLT-IIeA. The fusion protein purified by Glutathione Sephrose 4B as antigen immunized BALB/C mice. GST expressed in recombinant E.coli pGEX-6P-l as negative antigen and SLT-IIeA as positive antigen absorbed hi ELISA plate. SP2/0 myelomacells and spleen cells of the immunized BALB/C mice were fused by PEG-2000. The hybridoma culture superaatants were screened for anti-SLT-IIeA antibodies by indirect-ELISA and cytotoxicity neutralization. One monoclonal antibody A2-D3 was detected and only reacted with a 61KDa fusion protein band in Western-blot. The Mab as probe has detected 13 strains of ED by West-blot, which shows 13 strains were positive.The A subunit gene (fedA,510bp) of the F18ab fimbriae was amplificicated from Escherichia coli F107/86 by polymersae chain reaction (PCR).The PCR product of fed A was cloned into plasimid vector pGEX-6P-l at BamFfl and EcoRI sites.The gene was genetically inserted at the downsteam of the 3"terminus of the gene coding for enzymeglutathione S-transferase ,which served as a carrier in this expression system.Recombinant expression plasmid pF107 was constructed .The coding sequence of SLT-IIeA(sltA,900bp) was amplified from pSLT-HeA and was cloned into pF107 at EcoRI and Sail sites ,the resulting recombinants was called pFSLT- II eA. The result of SDS-PAGE and Western-blotting assay demonstrated that the fusion protein(GST-F18ab-SLT-IIeA) expressed by the recombinant plasmid pFSLT-HeA can specifically respone to the monoantibody against F18ab fimbriae and SLT-IIeA.This indicated that the expected co-expression protein was successfully obtained.ICR mice were immunized twice with pF107, pSLT-HeA, pFSLT- II eA respectively and the immunized sera were toxined with E.coli F107/86 which express fimbriae F18ab and SLT-IIe.The result shows that the coexpression protein can protect the mouse.