Prokaryotic Expression And Purification Of C. Difficile-GDH And Development Of Colloidal Gold Strip For Detection Of The Bacterium

Clostridium difficile is the main pathogen causing antibiotic associated diarrhea,and also the diarrhea in the hospital.As for CDI diagnosis,firstly,GDH was screened by ELISA,and the positive cases were detected by cytotoxin neutralization test for toxin test.There is no epidemiological investigation report on the CDI in China and no approved commercial detection kit.In this experiment,glutamic dehydrogenase was expressed and purified,and the antigenic polyclonal antibody was prepared by immunizing the rabbit.After purification,polyclonal antibody was used to establish a colloidal gold immunochromatographic assay for GDH.It provides a new and highly specific detection method for CDI's clinical detection and epidemiological investigation.PET30a(GDH)expression plasmid was constructed with GDH complete sequence which cloned by PCR method,then transmute the plasmid into E.coli BL21 strain to express the glutamate dehydrogenase(GDH).The recombinant protein was used as antigen to prepare polyclonal antibody by immunizing the rabbit.Subsequently,purified GDH polyclonal antibody was used as immune gold antibody,and GDH monoclonal antibody was used as the T-lineantibody to established the Clostridium difficile-GDH colloidal gold immunochromatographic assay.After successful expression of GDH protein through the prokaryotic expression system,protein band size in line with expectations which was about 51 kDa.What's more,the purity of GDH purified by Ni-NTA system can reach more than 90%.Then the purified protein was immunized rabbits and get polyclonal antibody of titer 1:51200.Moreover,the colloidal gold strip developed with polyclonal antibody has been tested with specificity and no sensitivity to other intestinal bacteria.The sensitivity is 80ng/mL and the accuracy rate was 80.36% by clinical test and compared with the PCR method.In conclusion,the GICA for CD that established in this study has good specificity and accuracy,providing a new idea for the clinical diagnosis of CDI.