Cloning Of The 3'-terminal Repeat Domain Of Clostridium Difficile Toxin A By PCR And Its Clinical Significance

Background/Objectives: Clostridium difficile is a major agent of antibiotic-associated disease(AAD) and pseudomembranous colitis(PMC). A number of large outbreaks of C. difficile-associated disease have been reported. The incidence of antibiotic-associated colitis has increased with increasing use of broad-spectrum antibiotics, multiple antibiotics, and antineoplastic drugs with antibiotic properties. Antibiotics or antineoplastid drugs suppress normal gut flora, allowing overgrowth of Clostridium difficile. Endogenous or exogenous toxingenic clostridium difficiles reside in gut, producing two toxins( A and B), which cause colonic mucosal inflammation. The therapy for AAD may require discontinuing antibiotics and intravenous fluids. In more serve case, the enteric route of therapy is preferred, because C. difficile remains within the colonic lumen without invading the colonic mucosa. Vancomycin and metronidazole are antibiotics generally used to treat AAD and PMC. But with the much more relapse(>25%) and increasing morbidity and mortality, it's necessary to research the immmunity and gene bacterin treatment. Otherwise, the old testing of infection of CD is taken time and strenuosity. The diagnose with gene probe is a new advance method. C. difficile produces 2 toxins, both of which are important to the pathogenesis of AAD. Toxin B is a potent cytotoxin, thereas Toxin A is an enterotoxin and ctotoxin likely to be responsible for the colitis that allows Toxin B to enter the cell. In recent years, Toxin A is becoming the research key. The gene coding Toxin A protein includes 8407bp. There are many of repeat units in if s 3' terminal. These repeat units is proved to code conglutinating protein. So we choose Toxin A as a target to obtain a genie fragment from 3' terminal and make a analysis. On the bases we can go on the other research such as treatment or clinical testing.Method: (1) Collecting liquid or unformed stool specimens and isolating CD with CCFA and CCAA in absolute anaerobic conditions, collating with VPI10463. All bacilli are indentified with API system. (2) Distilling genome DNA as template, amplify genie fragment by PCR from template. Sequencing and making analysis with Gel. (3) Making prediction about protein.Results: (1) We gained 2 toxingenic C. difficile from 4l stool specimens and indentified with API system. (2) We success to clone the gene fragment from the COOH- of CD. The results of sequence is same to GenBank with 959 base pairs and 2 ORF, coding 318aa. (3) Secondary structure prediction indicate that this peptide conclude many hydrophobicity baseline; The fragment contained a short sequence of amino acids(A'STDYGK) which is the putative receptor-binding posoitions.Conclusion: (1)\ We have established a series of stable way of isolating C. difficile through our trial and validated the new CCAA. (2^ A shortcut way to distill genome DNA may be used in clinical testing. (3)> We successed to obtained a new gene fragment from the 3'-terminal of toxin A gene, which has 950 base pairs and are conservative in C. difficile genome DNA. (4)> The gene fragment we gained coded a peptide. This peptide has a porperty of hydrophobicity . A possible epitopes may exist in the peptide. We can go on the research of vaccine by genenic engineering technique.