Development of recombinant strain producing C. perfringens beta, epsilon and C. novyi alpha toxins for use in combined clostridial vaccine production

Enterotoxemia and infectious necrotic hepatitis are important bacterial diseases caused by Clostridium species, causing high mortality rates and significant economic losses in small ruminants. Enterotoxemia is generally associated with toxins of C. perfringens type C and D strains, while infectious necrotic hepatitis is associated with toxins of C. novyi type B. Vaccines and hygiene/nutritional measures have traditionally been used to control these diseases. However, difficulties in the production of conventional vaccines, including the inability of toxins to sufficiently stimulate the immune system, have increased the need for more effective and safe vaccine strategies. Therefore, modern approaches such as recombinant DNA technology and synthetic biology enable high-throughput and scalable vaccine production with defined antigens. In this thesis, the design and construction of recombinant E. coli strains were conducted for use in the development of an effective vaccine candidate against enterotoxemia and infectious necrotic hepatitis. Seven different plasmid-mediated strains carrying the target toxin genes, namely "pCpDetx, j23119-pelB-CpDetx, pET-28(a)-pelB-CpDetx, j23119-pelB-CpCcpb, pET-28(a)-pelB-CpCcpb, j23119-pelB-CnBtcn-alpha, pET-28(a)-pelB-CnBtcn-alpha", were developed. While constructing these plasmids, higher levels of protein expression were aimed by incorporating additional gene sequences into the design. In addition, the plasmids were tested in host systems capable of high-level toxin production (e.g., E. coli BL21(DE3), C41(DE3), C43(DE3)), and high protein expression was observed in the E. coli C43(DE3) strain. In addition to the plasmid-mediated construction of the CpDetx and CpCcpb toxin genes, their integration into the genome, both individually and in pairs, was also successfully completed. In this way, genome-integrated strains designated "E. coli cpb+, E. coli etx+, and E. coli etx+ cpb+" were obtained. The expression levels of the produced recombinant toxin proteins were analyzed by SDS-PAGE, and the optimal plasmid and host combination was determined by comparing band intensities. According to the results, the E. coli C43 (DE3) strain carrying the pET-based expression plasmid (pET-28(a)-pelB-CpDetx, pET-28(a)-pelB-CpCcpb, pET-28(a)-pelB-CnBtcn-alpha) provided high levels of protein expression for the toxin genes.