Preparation Of Monoclonal Antibodies Against EHEC O104:H4 And Development Of Rapid Gold Immunochromatography Assays For EHEC O104:H4 Detection

??Background and ObjectiveA large-scale EHEC O104:H4(Enterohemorrhagic Escherichia coli,EHEC)outbreak began in German,and quickly spread to 16 countries,including Europe,America and other countries in May 2011.A total of 4137 cases(50 deaths)were reported worldwide.EHEC O104:H4's high pathogenesis had a higher relatedness to EAEC?EHEC and EPEC.EHEC O104:H4 had the following virulence genes:shiga toxin 2(stx2)?regulatory gene of plasmid(aggR)?transporter protein gene(aatA)?fimbriae gene(aggC)and fimbral gene(aggA).EHEC infection could cause bloody diarrhoea?haemorrhagic colitis?haemolytic uraemic syndrome and thrombotic thrombocytopenic purpura.AatA was encoded by pAA virulence plasmid.The aatA gene produced a outer membrane protein that was part of the aat-PABCE transportation system and was a main pathogenic gene of EHEC 0104:H4.The structure of aatA gene was comprised of three domains:an amino-terminal signal peptide?a passenger domain,which had the function of the secreted protein and a C-terminal domain that mediates secretion through the outer membrane.EHEC pathogenicity was primarily correlated to the production of shiga toxin 2,with A1 segment was its virulence activity center.This study attempted to artificially synthesize aatA and stx2A1.The prokaryotic expression was carried out and monoclonal antibodies was prepared.Anti-GST-AatA and anti-GST-Stx2A1 monoclonal antibodies were developed colloidal gold test strip respectively.Test the sensitivity?specificity and repeatability of strips,verifying the performance.GST-AatA and GST-Stx2A1 double-target test strips were successfully prepared.It could detect EHEC O104:H4 strain,making 0104:H4 could be detected on-site testing.??Methods1.Gene cloning,expression and purification of EHEC O104:H4 aatA and stx2A1.The aatA and stx2A1 gene sequences were synthesized and connected to pMD18-T vector respectively,followed by the transformation of E.coli DH5?.The recycled PCR product was inserted into the prokaryotic plasmid PGEX-6P-1 and confirmed by DNA Sequencing.The constructed prokaryotic expression vector was used to transform E.coli BL21.IPTG was added to induce the expression of fusion protein.The purified protein was analyzed by SDS-PAGE and Western Blot.2.Preparation of monoclonal antibodies against EHEC 0104:H4 GST-AatA and GST-Stx2A1 protein.McAbs were generated by immunizing 7 weeks female Balb/c mice with 0.5 mL of GST-AatA protein in equal volume of Freund's complete adjuvant intraperitoneally two times.The mice spleen cells with anti-serum titer more than 1:10000 were fused to the murine myeloma.Hybridomas were suc.cessively selected using ELISA and limiting dilution.Ascitic fluid was generated by injecting hybridoma cells into Balb/c mice.McAb was purified with affinity chromatography.Indirect ELISA was used to detect ascites titer.3.Immunity colloidal gold detection technology's establishment.The sandwich GICA(Gold Immunochromatography Assay)to detect AatA and Stx2A1 protein were prepared.Immuno gold test strips were successful prepared to detect EHEC O104:H4.The specificity?sensitivity and repeatability were tested to determine the performence of these three test strips.??Results1.The expression of GST-AatA and GST-Stx2A1 proteinThe 510 bp aatA and 720 bp stx2A1 were obtained by PCR.The positive bands of 510 bp and 720 bp were obtained with E.coli BL21 as template.The recombinant protein GST-AatA and GST-Stx2A1 were successfully expressed in E.coli BL21.The molecular weight of the GST-AatA and GST-Stx2A1 protein were 46 kDa and 55 kDa.2.Preparation of monoclonal antibodies against EHEC 0104:H4 GST-AatA and GST-Stx2A1 protein.The antisera were prepared respectively by immunizing Balb/c mice with purified fusion protein GST-AatA and GST-Stx2A1.McAbs were purified with affinity chromatography.SDS-PAGE showed the purity of anti-GST-AatA and anti-GST-Stx2A1 monoclonal antibodies were 95%and 96%.3.Immunity colloidal gold detection technology's establishment.The sandwich GICA(Gold Immunochromatography Assay)to detect AatA and Stx2A1 protein were prepared.Immuno gold test strips were successful prepared to detect EHEC 0104:H4.??ConclusionThe 510 bp aatA and 720 bp stx2A1 were obtained by PCR.The positive bands of 510 bp and 720 bp were obtained with E.coli BL21 as template.The recombinant protein GST-AatA and GST-Stx2A1 were successfully expressed in E.coli BL21.Optimized GST-AatA and GST-Stx2A1 expression condition,we think that the expression conditions both were that E.coli BL21 was inducted by 1mM IPTG.The molecular weight of the GST-AatA and GST-Stx2A1 protein were 46 kDa and 55 kDa.The monoclonal antibodies were obtained by immunizing Balb/c mouse with purified GST-AatA and GST-Stx2A1 protein.The antibodies both were identified as IgG1.The purity of the fusion protein GST-AatA and GST-Stx2A1 were about 97%and 96.8%.Immuno gold test strips to detect EHEC 0104:H4 were successful prepared.