The genc CPA and ETX encoding α and e toxins were amplified from the standard strain C60-2 of type D Clostridium perfingens by PCR, and then they were inserted into pet-Duct-1 vecto, respectively for the construction...The genc CPA and ETX encoding α and e toxins were amplified from the standard strain C60-2 of type D Clostridium perfingens by PCR, and then they were inserted into pet-Duct-1 vecto, respectively for the construction of co-expression plasmid pETDoet-1-CPA-ETX. The co-expression plasmid was transformed into BL21 （DE3） competent cells, at and e toxins proteins were induced by IPTG before analysis by SDS-PAGE. As a result, both of α and e toxins were detected at 43 and 34 KU by western-blot. Mice immunized with the co-expressed α and e toxins proteins produced high titers of neutralizing antibodies in the serum, which protected the mice against the attack of type D C. perfringens culture filtrate. In addition, mice immunized with the produced co-expressed α and e toxins proteins showed significantly higher surviving rate than with ε toxins protein alone when infected with culture filtrate of type D C. perfringens . These results indicated that co-expression of α and e toxins proteins could be used as a new method to prepare vaccines against the pathogens of multiple serotypes.展开更多
Clostridium difficile is a Gram positive rod-shaped bacterium that produces two major toxins, A and B. The detection of the organism and its toxins has been widely carried out using specialized Enzyme-Linked Immunosor...Clostridium difficile is a Gram positive rod-shaped bacterium that produces two major toxins, A and B. The detection of the organism and its toxins has been widely carried out using specialized Enzyme-Linked Immunosorbent Assay (ELISA) kits;however these generally have been unsuccessful in identifying all Clostridium difficile positive samples. In this study, fifteen clinically symptomatic patients from three of the five major regional hospitals in Trinidad were investigated for Clostridium difficile infections. Stool samples were assessed by ELISA and cultured isolates were characterized using agar dilution antibiotic sensitivity assays, conventional Polymerase Chain Reaction (PCR), DNA sequencing and phylogenetic analysis of toxin A and B genes. All 15 patient stool samples and isolates were positive for toxigenic Clostridium difficile via ELISA and PCR respectively. All isolates were positive for the housekeeping tpi and Toxin B genes by PCR but only three of these were positive for the Toxin A gene. The Toxin B gene sequences showed 100% similarity levels among isolates while the Toxin A gene sequences showed 99% similarity among isolates. Phylogenetic analysis showed that the strains isolated in Trinidad most likely belonged to the same strain/group. Agar dilution sensitivity tests showed highest susceptibility to Pipercillin/Tazobactam and Meropenem (87%) and the highest resistance was seen with Cefotaxime in 93%. These results indicate that similar virulent strains of C. difficile are present in the Trinidad population and that pathogenic strains are more likely to be susceptible to Pipercillin/Tazobactam and Meropenem.展开更多
基金Supported by the Basic Scientific Research Project of Nonprofit Central Research Institutions(1610322013028)
文摘The genc CPA and ETX encoding α and e toxins were amplified from the standard strain C60-2 of type D Clostridium perfingens by PCR, and then they were inserted into pet-Duct-1 vecto, respectively for the construction of co-expression plasmid pETDoet-1-CPA-ETX. The co-expression plasmid was transformed into BL21 （DE3） competent cells, at and e toxins proteins were induced by IPTG before analysis by SDS-PAGE. As a result, both of α and e toxins were detected at 43 and 34 KU by western-blot. Mice immunized with the co-expressed α and e toxins proteins produced high titers of neutralizing antibodies in the serum, which protected the mice against the attack of type D C. perfringens culture filtrate. In addition, mice immunized with the produced co-expressed α and e toxins proteins showed significantly higher surviving rate than with ε toxins protein alone when infected with culture filtrate of type D C. perfringens . These results indicated that co-expression of α and e toxins proteins could be used as a new method to prepare vaccines against the pathogens of multiple serotypes.
文摘Clostridium difficile is a Gram positive rod-shaped bacterium that produces two major toxins, A and B. The detection of the organism and its toxins has been widely carried out using specialized Enzyme-Linked Immunosorbent Assay (ELISA) kits;however these generally have been unsuccessful in identifying all Clostridium difficile positive samples. In this study, fifteen clinically symptomatic patients from three of the five major regional hospitals in Trinidad were investigated for Clostridium difficile infections. Stool samples were assessed by ELISA and cultured isolates were characterized using agar dilution antibiotic sensitivity assays, conventional Polymerase Chain Reaction (PCR), DNA sequencing and phylogenetic analysis of toxin A and B genes. All 15 patient stool samples and isolates were positive for toxigenic Clostridium difficile via ELISA and PCR respectively. All isolates were positive for the housekeeping tpi and Toxin B genes by PCR but only three of these were positive for the Toxin A gene. The Toxin B gene sequences showed 100% similarity levels among isolates while the Toxin A gene sequences showed 99% similarity among isolates. Phylogenetic analysis showed that the strains isolated in Trinidad most likely belonged to the same strain/group. Agar dilution sensitivity tests showed highest susceptibility to Pipercillin/Tazobactam and Meropenem (87%) and the highest resistance was seen with Cefotaxime in 93%. These results indicate that similar virulent strains of C. difficile are present in the Trinidad population and that pathogenic strains are more likely to be susceptible to Pipercillin/Tazobactam and Meropenem.
