| Clostridium difficile,a strictly anaerobic,spore forming and gram positive bacillus,is considered to be the main cause of the antibiotic-associated diarrhea prevailing in hospital settings.In the past decade,the morbidity and mortality of C.difficile infections?CDIs?has increased significantly due to the abuse of antibiotic and the emergence of hypervirulent strain 027/NAP1/BI.Moreover,it seems to be less responsive to the traditional treatments.Therefore,the control,prevention,diagnosis,and therapies of CDIs become a public health concern worldwide.Toxin A?TcdA?and Toxin B?TcdB?,the two exotoxins of C.difficile,are the major virulence factors of CDIs,and TcdB is considered to be essential for CDIs.Given that the toxins play critical roles in the CDIs,current studies focus on the molecular mode of action of the toxins and the relationship of the toxins' structure and function and most of the diagnostic methods of CDIs rely on detection of C.difficile toxins by using an enzyme immunoassay.In this work,the essential region for the translocation of TcdB was identified and anti-TcdB monoclonal antibodies that can be used in the diagnosis of CDIs were prepared and evaluated.Firstly,TcdA and TcdB are both consist of four distinct domains,the glucosyltransferase domain?GTD?,cystein protease domain?CPD?,translocation domain?TD?and receptor binding domain?RBD?.Few studies have investigated the TD and its mechanis of action.Recently,it was demonstrated that a segment of 97 amino acids?D97?within the translocation domain of TcdB is essential for the in vitro and in vivo toxicity of TcdB.In this study,the secondary structure of the D97 was predicted by the algorithms GOR4,SIMPAA96 and Chou-Fasman?Prot Scal?and a smaller fragement AA1756-1780,located in the N-terminus of the D97,was found to be essential for translocation of the toxins.Using molecular cloning technique,deletion mutant TcdB?1756-1780 was constructed and expressed in the Bacillus megaterium system.After purification,the in vitro and in vivo toxicity of TcdB?1756-1780 was examined.The result showed that the in vitro and in vivo toxicity of TcdB?1756-1780 dramactically decreased by 1×106 fold,indicating that the region AA1756-1780 is critical for TcdB toxicity.To investigate the function of the region AA1756-1780,the overall structures of the holotoxin TcdBfl and TcdB?1756-1780 were analyzed by circular dichroism spectrophotometry,and the function of the four domains of toxin were examined.As the result,TcdB?1756-1780 maintained a similar secondary structure to TcdBfl and showed similar glucosytransferase and cystein protease activity,cellular binding,pore formation and conformational change pattern to TcdBfl.However,TcdB?1756-1780 failed to induce the glucosylation of Rho GTPase Rac1 of host cell.To obtain further insight into the behavior of the toxins in endosomes,endosome isolation and western blot analysis were performed,the result indicated that TcdB?1756-1780 was trapped in the endosomes and was subsequently degraded into small fragments,leading to the failure in GTD delivery.We hypothesize that the region AA1756-1780 has a critical role in p H-induced conformation change,and its deletion may lead to an incorrect conformational change,resulting in unsuccessful membrane insertion and GTD delivery.Secondly,because of the poor clinical different between CDIs and other causes of hospitalacquired diarrhea,laboratory test for C.difficile is an important intervention for diagnosis of CDIs.Toxin EIA has been the most frequently used assay in laboratory diagnosis for its low cost,rapid and ease to perform.However,the low sensitivity of toxin EIA makes it not adequate to be used as standalone tests and more sensitive antibodies are needed for enhance the sensitivity of the toxin EIA.Using the mutant toxin a TcdB as antigen,whose in vitro and in vivo toxicity has been lost,the mouses were immunized for four times before cell fusion,after which,sub cloning was performed and four clones of positive hybridoma A7,B9,D3 and G2 were selected and purified.The titer and affinity of the purified m Abs were determined by indirect ELISA and SPR assay respectively.The titer of A7,B9,D3 and G2 were 1×106,1×106,1×106 and 1×107,while the affinity constants were 4.29×10-9,1.01×10-7,1.22×10-8 and 1.84×10-11.For antibodies pairing using sandwich ELISA,the four m Abs were labeled with HRP and three pairs of antibodies G2-A7,G2-B9 and G2-D3 were selected.In order to develop an immunochromatography assay for TcdB detection,the m Abs A7 and G2 were applied to immunochromatography test strips.40 positive and 50 negative clinical stool samples were used for evaluation of the test strip.The specificity and sensitivity of the G2-A7 immunochromatography test strip are 90%?95% CI: 78.2%-96.7%?and 92.5%?95% CI: 79.6%-98.4%?respectively. |
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