Cloning And Expression Of The C-terminal Domain Gene Of Clostridium Difficile Toxin B Mucosa Receptor Binding Domain

Clostridium difficile was one major pathogen of nosocomial intestinal infections. It cause Clostridium difficile -associated disease (CDAD)in humans. Nowadays the predominant antibiotics for treatment of CDAD are metronidazole and voncomycin .However, clostridium difficile has been found to possesses multiple-antibiotic resistance genes. The CD 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. The aim of this study is to search a candidate for the vaccine of CDAD prevention and treatment through gene recombination technology .According to the feature of CD toxin B, the C-terminal domain of Clostridium difficile Toxin B (CDB3) gene was amplified by PCR techniques with the whole genome DNA of the standard strain VPI 10463 as template. The PCR product was inserted into the prokaryotic expression vector pGEX-4T-1 after they are both digested by EcoR I and BamH I and linked by T4 DNA ligase . The recombinant plasmid was then transformed into the E.coli BL21(DE3) after sequencing and comparing with the standard sequence. Clostridium difficile Toxin B gene was expressed with the inducement of IPTG. The expression products were analyzed using the SDS-PAGE and Western blot method.Results1. CD was cultured successfully, and the genome was extracted as template of PCR reaction.2. The C-terminal domain of Clostridium difficile Toxin B gene about 1848 bp was amplified by PCR techniques .The primer was ideal which shown high specificity in the reaction .The PCR product was sequenced and compared with the standard sequence .It was confirmed that the PCR product was CDB3 gene.3. The recombinant vector was obtained after the CDB3 gene and express vector were linked .The gene segment inserted into recombinant vector was identified 3 00% to the Gene Bank.4. The fused protein was obtained by induced with IPTG. It was shown the expression product of the CDB3 gene with a molecular mass of 97.7 kDa by the detect method of 8% SDS-PAGE .The result was the same as expect.5. Western-blot shows the fused protein has been expressed successfully, which might act as a necessary basis for the further research work on the clostridium difficile diagnose reagent and vaccine.