Preparation And Primary Application Of Monoclonal Antibody Against B Subunit Of Escherichia Coli Type ? Heat-labile Enterotoxin

Escherichia coli-induced diarrhea is caused by E.coli in travelers,children and young animals,the most common pathogen is Enterotoxigenic Escherichia coli(ETEC).The enterotoxin produced by ETEC can result in severe diarrhea in piglets and calves by causing ion disorder and watersodium retention in the intestinal tract,causing huge economic losses to the cultivation industry.The main enterotoxins that play a pathogenic role in ETEC are heat-stable enterotoxin(ST)and heat-labile enterotoxin(LT).Recently,ETEC which produces type ? heat-labile enterotoxin(LT-?),has been isolated from calves with bacterial diarrhea,the classification of LT-? subtypes is complicated,the infection and the prevalence status between humans and animals shows an increasing trend year by year.However,the detection method for LT-? is relatively simple and its B-subunit has similar immune adjuvant effects to that of LT-?.Therefore,in-depth study of the immunogenicity and detection method of LT-? is significant for the prevention and treatment of diarrhea caused by E.coli.In this study,type ? heat-labile enterotoxin produced by ETEC was used as the research object.We designed primers to amplify gene segments of LT-? protein B subunit without the signal peptide.The gene fragments were double digested and ligated into p GEX-6p-1 and p MAL-c4 x expression vectors.After transformation,recombinant E.coli BL21/p GEX-6p-1-LT-?-B and BL21/p MAL-c4xLT-?-B were obtained for stably expressing the recombinant protein.The recombinant protein r GSTLT-?-B(38 KDa,inclusion body expression)and recombinant protein r MBP-LT-?-B(61 KDa,soluble expression)were obtained with optimized expression and purification conditions,respectively.Western blot analysis demonstrated that the two recombinant proteins could react with GST polyclonal antibodies and MBP polyclonal antibodies,respectively.The recombinant proteins have a single band after affinity chromatography,and the purpose of purification has achieved.BALB/c mice were immunized by subcutaneous injection on back with r MBP-LT-?-B which is fully emulsified with freund's complete adjuvant,the spleen cells from immunized mouse were fused with myeloma cells SP2/0.The fused cell lines were screen by indirect ELISA method.After subcloning,three hybridoma cells stably secreting monoclonal antibodies against LT-?-B were obtained,named 1C10,2A10 and 4D4,respectively.The potencies of antibodies produced by the hybridoma cells were stable after continuous passages.The light chains of the three monoclonal antibodies were identified by enzyme-labeled antibodies as ? chains.The heavy chains of MAb-1C10 and m Ab-4D4 were identified as Ig G1 subclass while the heavy chain of m Ab-2A10 was identified as Ig G2 b subclass.The supernatant potencies of hybridoma cells were from 1:800 to 1:3200,and the potencies of ascites were from 1:6 400 to 1:102 400.Western blot demonstrated that each monoclonal antibody could react specifically with r GST-LT-?-B.The detection results of the blocking ELISA showed that the crude LT-? natural toxin could block the binding of the three monoclonal antibodies to r GST-LT-?-B,while the blocking rates of LT-?,STa,Stx2 e,Stx1 and Stx2 toxins were lower than 50%,indicating that the three monoclonal antibodies did not react with other toxins and had good specificity.Neutralization experiments using Y-1 cells showed that 1C10,2A10 and 4D4 monoclonal antibodies all had neutralizing activity,the neutralizing titers are 1:71,1:22,and 1:40,respectively.In order to accurately identify the LT-?-B antigenic epitopes recognized by each monoclonal antibody,9 synthetic peptides covering the whole LT-?-B protein were coated as antigens respectively,and the prepared monoclonal antibodies were used as primary antibodies,the recognition sites of different monoclonal antibodies were identified by indirect ELISA method.Finally,it was determined that the epitope recognized by m Ab-1C10 was 51-GGQYYPDNYL-60,the epitope recognized by m Ab-2A10 was 31-NKDSK-35,and the epitope recognized by m Ab-4D4 was 86-YTPNHVWAIELAP-98.The spatial structure analysis of antigenic epitopes using Protean software and online website showed that the epitopes recognized by each monoclonal antibody were located in the region with high hydrophilicity and antigenic index,with a curved linear structure.The three epitopes have no homology with LT-?,31-NKDSK-35 epitope is highly conserved among the 6 subclasses of LT-?c,it has no homology with LT-?a and LT-?b;51-GGQYYPDNYL-60 epitope and 86-YTPNHVWAIELAP-98 epitope are highly conserved in LT-?c1 and LT-?c5,one to two amino acid mutations exists in LT-?c2,LT-?c3,LT-?c4 and LT-?c6,these two epitopes have low homology with LT-IIa and LT-IIb.A blocking ELISA method for the detection of LT-? was initially established using r GST-LT-?-B as the coating antigen and m Ab-1C10 as the detection antibody.The reaction program of each step was optimized on the premise of the highest blocking rate.The results showed that the coating concentration of antigen was 1 ?g/m L,the optimal dilution of the detection antibody m Ab-1C10 was 1:800,the optimal working concentration of HRP-Ig G was 1:10 000,the optimal incubation time of secondary antibody was 30 min,the optimal sealing time,blocking time and reaction time of substrate were 2 h,1 h 30 min and 15 min,respectively.The cut-off value of blocking rate is53.15%,the results of specificity and repetitive tests illustrated that the method had good specificity and repetitive,and the minimum detection amount of crude LT-? toxin was 1 mg/m L.In summary,we expressed the r MBP-LT-?-B with immunogenicity and the r GST-LT-?-B for antibody detection and immune monitoring.On this account,three hybridoma cell lines,1C10,2A10 and 4D4,which stably secrete high potency antibodies were prepared and screened.All three monoclonal antibodies have neutralizing effects on natural LT-? toxin.A blocking ELISA method for the detection of LT-? was initially established with the 1C10 monoclonal antibody,and the antigenic epitopes of LT-?-B recognized by three monoclonal antibodies were identified,which laid the foundation for the detection of LT-?,the development of epitope vaccines and the research of new adjuvants.