Cloning of Immunogenic Domain of Clostridium Difficile Toxin B in Lactococcus lactis to Develop an Oral Vaccine Based onLactococcus against Clostridium difficile Associated Colitis

Clostridium difficile bacterium is the leading cause of clinical infection and pseudomembranous colitis. Toxin B (TcdB) of C. difficile is regarded as one of the main virulence factors of this bacterium. Moreover, the receptor binding domain (RBD) of this toxin which is known as an immunogenic domain attaches to its receptor in intestine. This study aimed to clone and express the immunogenic domain of the Toxin B in Lactococcus lactis to provide a safe oral vaccine against colitis caused by C. difficile as an alternative treatment in future studies based on Lactococcus.

In order to clone this segment, at first, the DNA was extracted from C. difficile, and then, the RBD gene segment was amplified by the PCR method using designed primers. The amplified segment was attached to pTZ25R/T plasmid and transformed into Escherichia coli. Subsequently, the RBD segment was extracted from pNZ9418 plasmid using restriction enzymes, such as SacI and NcoI, and then it was purified in this study. Following that, the RBD segment was cut in pNZ8149 plasmid using the same enzymes and was attached under the proper condition. The pNZ8149 plasmid with segment was transformed into L. lactic competent cells by electroporation method.

After the screening of electro-transformed colonies, the accuracy of cloning was verified using PCR, enzyme digestion on the extracted recombinant plasmid, and sequencing.

According to the results, L. lactic which produces the immunogenic domain can be used as a safe oral vaccine and can be considered as an alternative therapy to reduce the incidence of Clostridium difficile infection in further investigations.