In silico fusion of epsilon and beta toxin genes of Clostridium perfringens types D and B

Fusion protein technology represents the strategy to achieve rapid, efficient, and cost-effective protein expression. Epsilon and Beta toxins are the most potent Clostridial toxins and cause disease in animals. This study describes in silico fusion of Clostridium perfringens types D and B epsilon and beta toxin genes that was used for cloning in E. coli. The etx and cpb genes were retrieved from the GenBank and a fusion gene was designed to produce a chimeric fusion protein. Secondary and tertiary structures and specificities of fusion protein were determined by online software. Results showed that the designed fusion gene construction is suitable for chimeric fusion protein expression

Cloning and expression of S1 subunit of pertussis toxin in Escherichia coli

Bordetella pertussis is a gram negative bacterium that causes respiratory tract infection in human [whooping cough]. Pertussis toxin [PT] is the main component of current acellular pertussis vaccine and the S1 [subunit1] is the main immunogenic part of it. Thus, S1 has been the target of many studies as a potent candidate of acellular vaccine against Bordetella pertussis, lacking the side effects of whole cell based ones. S1 gene was amplified and inserted in three expression vectors including pET-14, pET-22b[+] and pAED4. The possibility and level of expression of these constructs were investigated in BL21 [DE3] strain of Escherichia coli [E.coli] as expression host. The highest expression was in pET-22b[+]-S1. Best expression achieved 6 hr post induction with 0.2 mM IPTG in LB broth containing ampicillin, at 30°C with shaking [250 rpm]. Recombinant S1 protein was observed in two distinct separated proteins with 28 and 31 kDa estimated molecular weight. In spite of toxicity of PT and S1 in the E.coli, considerable amount of S1 was expressed in E.coli. Two rS1 bands were detected on SDS-PAGE. Both were confirmed as S1 in western blot with specific monoclonal and polyclonal antibodies against pertussis toxin. Appearance of two distinct bands could be the result of leader peptidase activity or nonspecific peptidase from E.coli on recombinant S1. As the recombinant S1 is a suitable antigen for studies as a candidate acellular vaccine or development of ELISA for detection of Bordetella pertussis, further studies are underway