The Preparation And Preliminary Application Of Monoclonal Antibodies Against Clostridium Difficile TcdB Receptor Binding Region

Clostridium difficile(C.difficile)is a conditional pathogen that is widely found in the environment and various animals and can cause diseases such as diarrhea and pseudomembranous colitis.It is a serious threat to human health and safe production in the aquaculture industry.ST11 type C.difficile is currently one of the most widespread and most harmful subtypes in the international arena.Therefore,this study aimed to express the ST11 type C.difficile Tcd B toxin protein receptor binding region(RBD),screen for a monoclonal antibody against ST11 type Tcd B RBD,and use this antibody to initially establish a double for detecting ST11 type C.difficile Tcd B toxin.Antibody sandwich ELISA method.First,recombinant E.coli capable of expressing the ST11 type Tcd B RBD segment was successfully constructed.E.coli BL21(DE3)was used as the expression vector,p ET-32a(+)was used as the expression vector,and the Tcd B RBD(1848 bp)gene fragment was inserted into the vector.After 4 hours of induction with IPTG at a final concentration of 1 m M,the recombinant E.coli could express about the size.Recombinant RBD protein of about 97 KD.Secondly,hybridoma cells capable of secreting monoclonal antibodies against ST11 type Tcd B RBD were successfully screened.After immunization with recombinant RBD protein,the antibody titer reached1:51200.A hybridoma-positive hybridoma cell AE2D3 was obtained by hybridoma technique.The subtype identification result was Ig G2b(κ),and octanoic acid-sulfate was used.Ascites was purified by ammonium crude extraction and Protein G gel affinity chromatography.The purity was above 90% and the concentration was 0.743 mg/ml.It was confirmed by Western blot to be an anti-RBD specific antibody.Finally,a double antibody sandwich ELSIA method for the detection of ST11 type C.difficile Tcd B toxin was successfully established.The optimal reaction conditions were 0.05 m M carbonate coated with 1:1600 polyclonal antibody at 37 ℃ for 1 h + 4 ℃ overnight incubation,3% skim milk was blocked at 37 ℃ for 1 h,and the sample to be tested was added and AE2D3 detection antibody was added at 0.23 ug 37 ℃ for 1 h.Finally,the HRP enzyme-labeled secondary antibody was added for 0.5 min after incubation at 37 ℃ for 10 minutes.Using this method to detect clinically isolated ST11 type C.difficile positive rate was 100%,and other control samples were all negative.The detection method has high specificity and good sensitivity.