Gene Cloning, Expression And Its Biological Characteristic Of Clostridium Difficile Toxin A Mucosa Receptor Conjugate Region

Clostridium difficile, a spore-forming anaerobe, is the predominant pathogen of nosocomial intestinal infections, especially the elderly and the infant, who suffer from the antibiotics associated diarrhea(AAD) to pseudomembramous colitis(PMC) for being treated with antibiotics or chemical therapy for a long time. This organism causes about 25% of the cases of AAD, up to 75% of the cases of antibiotic associated colitis, and virtually all the cases of PMC. Antibiotics or some cancer chemotherapeutics suppress normal gut flora, allowing overgrowth of clostridium difficile . The major detection of CD is to culture the patients' stool with CCFA medium, then test the cytotoxicity of the culrure(golden standard to detect CD). This method takes much more time and expense. Another method to detect CD is using ELISA to detect the toxin A of CD (cdtA ) , this method has low sensitivity and relatively high false negative. Nearly all antibiotics, including vancomycin and some cancer chemotherapeutics, can induce Clostridium difficile-associated disease (CDAD). So, even antibiotic treatment is problematic for use in treating CD AD. Nowadays the antibiotics of choice for treatment of CDAD are metronidazole for mild to moderate cases and vancomycin for moderate to severe cases. Although most patients respond to metronidazole or vancomycin, approximately 20% of patients relapse 2 to 8 weeks after the discontinuation of antibiotic therapy, moreover clostridium difficile has been found to possess multiple-antibiotic resistance genes , especially C. difficile clinical isolates resistant to both vancomycin and metronidazole have been reported , that means these drugs will become invalid in the future. It is necessary to investigate the vaccine to treat CDAD. Our aim of this study is to search a candidate for the vaccine of CDAD prevention and treatment and find a quick and simple method to detect CD.Methods:1. To amplify the 3' terminal gene of cdtA then ligate it with the plasmid pET-22b(+) after they are digested with EcoR I and Xho I for two hour, then transfer the recombined gene to E. coli BL21 ( DE3 ) . To induce the E. coli BL21 with IPTG, obtain the recombined protein. The purified recombined protein was tested its toxiciry by flow cytometry(FCM) and cytotoxicity assay, its antigenicity by western-blot assay.2. Using PCR To amplify the 3' terminal gene of cdtA and cdt B which code the membrane receptor conjugated protein to test the patients' stool, anaerobic culture the patients' stool at the same time with CCFA medium, then proceed biochemical analysis with the culture. The results were X2 analyzed with SPSS 10.0.Results:1. We cloned and expressed the membrane receptor conjugated protein of cdtA, FCM shows that the recombined protein has non-toxicity and protect the epithelial cells. Western-blot shows it has strong antigenicity.2. PCR is superior to bacterium culture of CD detection (p<0.05).The coincidence rate of the PCR and bacterium culture is 100%.Conclusion:1. We cloned and expressed the membrane receptor conjugated protein of cdtA, and confirm that this protein has strong antigenicity and non-toxicity. This protein can act as gene engineering recombined vaccine, and it also can be used as antigen for CD detection.2. We used PCR to detect the CD, which is superior to bacterium culture, and biochemical assay after bacterium culture. The coincidence rate of the PCR and bacterium culture is 100%. For PCR detection save time and money ,it can used widely for CD detection.