[Cloning of Clostridium perfringens alpha-toxin gene and extracellular expression in Escherichia coli].

Clostridium perfringens (C. perfringens) is a Gram-positive bacterial pathogen that widely propagets in the soil, the sea, the liver and the gastrointestinal tract of human and animals. This bacteria causes food poisoning, gas gangrene and other various range of infectious diseases. But there is no standard diagnosis method of C. perfringens. Therefore, to develop a new type of immunoassay for clinical purpose, we tried expression and extracellular secretion of recombinant alpha-toxin having enzyme activity in E. coli expression system. Cloning was carried out after PCR amplification from C. perfringens GAI 94074 which was clinical isolate. Three kinds of fragment was cloned using pET100/D-TOPO vector. These fragments coded for ribosome binding site, signal peptide, and alpha-toxin gene respectively. Recombinant pET100 plasmid transformed into TOP 10 cells and the obtained plasmids were transformed into BL21 (DE3) cells. Then, the transformants were induced expression with IPTG.. The expressed proteins were purified by Ni-Sepharose affinity column. The purified proteins were identified by capillary electrophoresis and LC/MS/MS. INTRODUCTION Clostridium perfringens is commonly found in the gastrointestinal tract of both humans and other animals, as well as in soil and sewage. C. perfringens has been shown to be a cause of human disease such as gas gangrene (clostridial myonecrosis), food poisoning, necrotizing enterocolitis of infants, and enteritis necroticans. It is also the causative agent of animal diseases such as lamb dysentery, ovine enterotoxemia and pulpy kidney disease of sheep, and other enterotoxemic diseases of lambs and calves (Sakurai et al., 1995). Strains of the bacterium can be classified as types A, B, C, or D, depending on the spectrum of toxin produced, in that alpha-toxin was produced by all types (Awad et al., 1995). So, C. perfringens ranks among most important of the anaerobic bacterial pathigens for human and domestic animals, and its prevention and clinical treatment are a serious key-issue (Songer, 1996). But there is no standard diagnosis method. The aim of this study is to develop a new type of immunological diagnostic targeting alpha-toxin for clinical purpose. As immunological method need specific antibody, we attempted to make and purify the antigen (recombined alpha-toxin) with recombinant technology.