Development of a New Bifunctional Fusion Protein of Vaccine Strains Clostridium perfringens Type D and Clostridium septicum Epsilon-alpha Toxin Genes
Marzieh Kamalirousta
Department of Biology, Kharazmi University, Alborz, Karaj, Iran.
Reza Pilehchian *
Specialized Clostridia Research Laboratory, Razi Vaccine and Serum Research Institute, Agricultural Research, Education and Extension Organization (AREEO), Alborz, Karaj, Iran.
*Author to whom correspondence should be addressed.
Abstract
There are various difficulties regarding of produce and design of a suitable fusion protein, and functionality is the most important of this problem. For producing a functional fusion protein, it is necessary to have a proper suitable host to clone a properly designed fusion gene. Clostridium perfringens and C. septicum are important pathogens of humans and livestock and produce numerous toxins including epsilon and alpha, which are responsible for severe diseases. In this study, a new construct of C. perfringens type D epsilon and C. septicum vaccine strains alpha toxin genes designed in addition, cloned into a prokaryotic host. Online software used for in silico, study and the prediction of the fusion protein construct primary, secondary and tertiary structures. At the next step, specific primers used for amplification of these genes, based on nucleotide sequences that retrieved from GenBank. The amplified etx and CSA fragments linked together by a linker based on a helix forming peptide (i+4) E, K. The linker introduced between two domains by fusion PCR and using Pfu DNA polymerase, epsilon forward and alpha reverse primers. The fusion gene ligated into pGEM-B1cloning vector and cloned into E. coli strain TOP10. This epsilon-alpha fusion gene could be used for development of a recombinant epsilon alpha fusion protein vaccine.
Keywords: Anaerobes, bioinformatics, cloning, fusion PCR