Molecular Cloning of a New Bifunctional Fusion Protein of Clostridium perfringens Type A alpha and Clostridium septicum alpha Toxin Genes in E. coli.

Background and Objectives: Fusion protein technology represents the strategy to achieve rapid, efficient, and cost-effective protein expression. There are various difficulties regarding the produce and design of a suitable fusion protein, and functionality is the most important of this problem. Clostridium perfringens type A and Clostridium septicum produce numerous toxins including alpha (plc) and alpha toxins, respectively, which are responsible for severe diseases. C. perfringens causes anaerobic cellulitis, gas gangrene, enteritis necroticans, and food poisoning in humans and gastrointestinal and enterotoxemic diseases in other animals. Historically, C. septicum has played a significant role as a causative agent of traumatic gas gangrene. However, in recent years, C. septicum has become increasingly identified with non-traumatic gas gangrene in patients with various diseases that affect the colon. In the present study, a new construct of C. perfringens type A alpha (cpa) and C. septicum alpha toxin (csa) genes designed in addition, cloned into a suitable host. Materials and Methods: To produce chimeric fusion protein, the alpha-alpha fusion gene was designed according to nucleotide sequences of cpa(KY584046.1) and csa(JN793989.2) genes. Tertiary structure and validation of the fusion protein were determined by online software. The amplified cpa and csa gene fragments linked together by a linker A(EAAAK)2A. The linker was introduced between two domains by fusion PCR. The purified fusion gene was ligated into the pUC57cloning vector and cloned into E. coli TOP10. Results: Analysis of the alpha-alpha fusion protein, using the I-TASSER server showed: C-score= -3.17 and also Ramachandran plot analysis in order to validate the geometrical structure as a native-like protein. The sequence of the alpha -alpha fusion gene was deposited in the GenBank under accession number MK908396. 1% agarose gel electrophoresis of fusion PCR product and sequencing analysis showed DNA fragment length is ~2.3 kb. Screening gel electrophoresis (colony PCR) showed 996 bp length with our designed linker included. 1% agarose gel electrophoresis of extracted and purified recombinant plasmid (pUC557/αα) showed that the recombinant pUC557/αα is 5056 bp. The digested recombinant pUC557/αα revealed one 2346bp (our fusion gene) band and one 2710 kb (non-recombinant pUC57) band. Conclusion: To our knowledge, this is the first time that alpha-alpha fusion gene is designed and cloned into a suitable cloning vector. This alpha-alpha fusion gene could be used for the development of a recombinant alpha- alpha fusion protein vaccine. This synthetic construction also could serve as a model for the development and production of novel fusion protein for other potential proteins and toxins.