Inoue Masaharu, Kikuchi Maho, Komoriya Tomoe, Watanabe Kunitomo, Kouno Hideki
Advanced Research Center for Life Science and Human, Department of Applied Molecular Chemistry, Graduate School of Industrial Technology, Nihon University, 1-2-1 Izumichou, Narashino, Chiba 275-8575, Japan.
Rinsho Biseibutshu Jinsoku Shindan Kenkyukai Shi. 2007;18(2):127-35.
Clostridium perfringens (C. perfringens) is a Gram-positive bacterial pathogen that widely propagets in the soil 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. In order to develop a new type of immunoassay for clinical purpose, we studied 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 were 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. In conclusion, we successfully cloned, expressed and exteracellular secreted C. perfringens alpha-toxin containing signal peptide. Biologically, the obtained recombinant protein was positive for phospholipase C activity.
产气荚膜梭菌是一种革兰氏阳性细菌病原体，广泛存在于土壤以及人和动物的胃肠道中。这种细菌会引发食物中毒、气性坏疽以及其他各类传染病。但目前尚无产气荚膜梭菌的标准诊断方法。为开发一种用于临床的新型免疫测定法，我们研究了在大肠杆菌表达系统中具有酶活性的重组α毒素的表达及细胞外分泌情况。从临床分离株产气荚膜梭菌GAI 94074进行PCR扩增后进行克隆。使用pET100/D-TOPO载体克隆了三种片段。这些片段分别编码核糖体结合位点、信号肽和α毒素基因。将重组pET100质粒转化到TOP 10细胞中，然后将获得的质粒转化到BL21(DE3)细胞中。接着，用异丙基-β-D-硫代半乳糖苷（IPTG）诱导转化体表达。总之，我们成功克隆、表达并在细胞外分泌了含有信号肽的产气荚膜梭菌α毒素。从生物学角度来看，所获得的重组蛋白具有磷脂酶C活性。
