Preparation And Application Of Monoclonal Antibodies Against Enterotoxigenic Escherichia Coli F17 Fimbriae A And G Subunits

Diarrhea caused by Enterotoxigenic Escherichia coli（ETEC）is a digestive tract infectious disease of calves and lambs.The main clinical symptoms are severe diarrhea and dehydration.The disease can occur all year round,calves aged 2 to 3 weeks and lambs aged less than 6 weeks are susceptible.The disease has a high mortality rate and is mostly endemic or sporadic.Once the outbreak occurs,it will bring huge losses to the cattle and sheep breeding industry.ETEC is characterized by the ability to produce two types of virulence factors:adhesins,which promote colonization after binding to specific enterocyte receptors and enterotoxins,which promote fluid secretion.Adhesion,as the basis of ETEC infection of cells,is the prerequisite for determining whether the disease occurs.As the highly related fimbrial adhesin to calf and lamb diarrhea,the F17 fimbriae is mainly composed of structural subunit F17-A and adhesion subunit F17-G.The preparation of monoclonal antibodies（MAbs）,the identification of antigenic epitopes,and the establishment of detection methods for F17 fimbriae have great theoretical and practical significance for the in-depth study of the biological characteristics and pathogenic mechanism of ETEC with F17fimbriae,as well as the prevention and treatment of diarrhea caused by ETEC in calves and lambs.This study carried out the following research work:（1）In this study,the gene fragments of F17-A and F17-G after de-signal peptide were amplified in vitro,then digested with double enzymes and connected to p ET-32a and p GEX-6p-1 vectors and transformed into competent cells.After inducing their expression,the recombinant proteins r F17-A-His（34ku）,r F17-G-His（53ku）,r F17-A-GST（43ku）,and r F17-G-GST（62ku）were obtained.The recombinant proteins with the His tag were used as the immune proteins to immunize Balb/c mice,and the recombinant proteins with the GST tag were used as the detection proteins to detect the titer of the immunized mice.After cell fusion,screening and subcloning,three cell lines secreting MAb against F17-A were obtained,which were E9-A,A10-A,B11-A and three cell lines secreting MAb against F17-G were obtained,which were F2-G,C3-G,E5-G.The obtained 6 cell lines could stably secrete MAb,and the type of MAbs was Ig G1,and the light chain wasκchain.Among them,MAb A10-A and MAb F2-G have the highest titers,the supernatant antibody titers were 1:2¹²and 1:2¹¹,and the ascites titers were1:10⁹and 1:10⁶.MAb A10-A and MAb F2-G did not react with F4,F5,F6,and F41 fimbriae,showing good specificity.The results of the adhesion inhibition experiment showed that MAb E5-G could effectively inhibit ETEC（F17⁺）adhesion to intestinal epithelial cells.（2）In this study,the antigenic epitopes recognized by MAbs were identified by truncation expression method,peptide synthesis method and amino acid mutation method.The results of epitope identification of F17-A showed that the epitope region recognized by MAb E9-A was ⁴¹AKTAGLTPFT⁵⁰,the epitope region recognized by MAb A10-A was¹⁵¹TVNYTIVYQ¹⁵⁹,the epitope region recognized by MAb B11-A was ⁸¹TGNLTNTASS⁹⁰;The results of epitope identification of F17-G showed that the epitope region recognized by MAb F2-G was ¹AAVSFIGSTENDVGP¹⁵,the epitope region recognized by MAb C3-G was²¹¹GTTSLKLQCDAGVTV²²⁵,MAb E5-G recognized a conformational epitope involving aspartic acid（Asp）at position 89 of the lectin domain,and this aspartic acid is a key amino acid that affects the binding of this conformational epitope to adhesion receptors.The conservation analysis of epitopes showed that MAb A10-A could recognize all variants of F17-A,and MAb E5-G could recognize all variants of F17-G.（3）A bacterial adhesion cell model and an indirect immunofluorescence detection method were established by using the prepared MAb A10-A.The results showed that Caco-2cells were more conducive to the adhesion of F17 fimbriae than IPEC-J2 cells,and the adhesion effect was the best when the MOI=1:100 and adhesion for 1h.This model provides a research basis for exploring the adhesion mechanism of F17 fimbriae at the cellular level.The optimized conditions of indirect immunofluorescence detection method were:4%paraformaldehyde fixation,1%BSA sealing,the working concentration of primary antibody MAb A10-A was 1:200,overnight at 4℃or incubation at 37℃for 1 h,the working concentration of secondary antibody was 1:100,and the incubation time was 1 h.It has been verified that this method only produces specific fluorescence with F17 fimbriae,showing good specificity.Three strains of Escherichia coli with F17 fimbriae were preliminarily detected by using this method.The results showed that this method could identify the adhesion ability of different strains,and the difference was obvious,which was consistent with the results of fluorescence quantitative PCR（q PCR）,so this method can be used to detect the adhesion mechanism of F17 fimbriae in the laboratory.（4）An indirect agglutination detection method using nanospheres as the carrier was established by using the prepared MAb A10-A.The optimized conditions for microsphere sensitization were:410 nm diameter microspheres were 20μL,0.06 M acetate buffer was 500μL,the purified MAb A10-A was 15μg and EDC was 30μg,agglutination at 37℃for 2h,then the agglutination effect was the best.The results of specificity,sensitivity,repeatability,and stability identification showed that only bacteria with F17 fimbriae agglutinated,and the minimum detectable bacterial concentration was 10⁴CFU/m L,the identification results between intra-assay and inter-assay were consistent,and the allergenic microspheres storage at 4°C for 60 days did not affect the detection results,means the method has good specificity,sensitivity,repeatability,and stability.The method was used to detect bacterial samples,and the positive coincidence rate between the detection results and the PCR detection results was100%,so this method can be used for clinical detection of F17 fimbriae.In this study,by analyzing the structural characteristics of F17 fimbriae,monoclonal antibodies were prepared against the subunits with different functions,and the antigen epitopes recognized by them were identified,which provided an effective biological preparation for the establishment of detection methods for F17 fimbriae,and provided the reliable biological information for the study of the adhesion mechanism of F17 fimbriae and the development of new drugs and vaccines.The established indirect immunofluorescence detection method of bacterial adhesion and the indirect agglutination detection method of F17fimbriae antigen provided effective technical support for the pathogenic detection and epidemiological investigation of ETEC with F17 fimbriae.