Saito Hitoshi, Inoue Masaharu, Tomiki Masayoshi, Nemoto Hiroshi, Komoriya Tomoe, Kimata Junko, Watanabe Kunitomo, Kohno Hideki
Advanced Research Center for Life Science and Human, Department of Applied Molecular Chemistry, Graduate School of Industrial Technology, Nihon University, 1-2-1 Izumicho, Narashino, Chiba 275-8575, Japan.
Rinsho Biseibutshu Jinsoku Shindan Kenkyukai Shi. 2009;20(1-2):9-20.
Only a few methods exist for simple, sensitive and rapid detection of alpha-toxin in clinical and biological samples. The aim of our study was to establish a procedure for the production of an antibody against a recombinant antigen with confirmed sequence identity. We applied a noble approach based on proteomics using a mass spectrometer for the conclusive identification of the recombinant alpha-toxin that was subsequently used as an antigen. The recombinant alpha-toxin was produced in Escherichia coli. A clinical isolate of Clostridium perfringens GAI 94074 was amplified by polymerase chain reaction (PCR) and subsequently, cloning was performed. Three different fragments were cloned using a pET100/D-TOPO vector. These fragments coded for a ribosome binding site, a signal peptide and the alpha-toxin gene, respectively. Recombinant pET100 plasmids were cloned into TOP 10 cells and the isolated plasmids were transferred into BL21 Star (DE3) cells. Their expression was then induced with isopropyl-beta-D-thiogalactopyranoside (IPTG). Recombinant E. coli transformed with a plasmid encoding the alpha-toxin gene alone produced a biologically inactive protein. On the other hand, E. coli carrying the plasmid encoding the toxin sequence and its native signal peptide sequence, or the toxin sequence along with the ribosome binding sequence and the signal peptide sequence secreted an active alpha-toxin with phospholipase activity. Accordingly, the C. perfringens gene encoding the alpha-toxin protein along with its signal peptide was successfully cloned, expressed, and secreted by E. coli. Furthermore, without consideration of its activity, we used mass spectrometry to confirm that the expressed protein was indeed the alpha-toxin. Thus, the identification of alpha-toxin protein using both the biological activity testing and the mass spectrometry analysis is expected to verify the significant production of C. perfringens antibody. The study for the analysis of recombinant alpha-toxin using ESI/MS has not been reported. In this study, we report the successful cloning, expression, secretion, identification and sequence determination of the C. perfringens alpha-toxin.
目前，用于临床和生物样本中α毒素简单、灵敏且快速检测的方法仅有几种。我们研究的目的是建立一种程序，用于生产针对具有确认序列同一性的重组抗原的抗体。我们采用了一种基于蛋白质组学的新颖方法，使用质谱仪对重组α毒素进行最终鉴定，随后将其用作抗原。重组α毒素在大肠杆菌中产生。通过聚合酶链反应（PCR）扩增产气荚膜梭菌GAI 94074的临床分离株，随后进行克隆。使用pET100/D-TOPO载体克隆了三个不同的片段。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质粒克隆到TOP 10细胞中，并将分离的质粒转移到BL21 Star（DE3）细胞中。然后用异丙基-β-D-硫代半乳糖苷（IPTG）诱导其表达。仅用编码α毒素基因的质粒转化的重组大肠杆菌产生了无生物活性的蛋白质。另一方面，携带编码毒素序列及其天然信号肽序列，或毒素序列以及核糖体结合序列和信号肽序列的质粒的大肠杆菌分泌出具有磷脂酶活性的活性α毒素。因此，产气荚膜梭菌编码α毒素蛋白及其信号肽的基因成功地被大肠杆菌克隆、表达和分泌。此外，不考虑其活性，我们使用质谱法确认表达蛋白确实是α毒素。因此，使用生物活性测试和质谱分析鉴定α毒素蛋白有望验证产气荚膜梭菌抗体的大量产生。尚未有关于使用电喷雾电离质谱（ESI/MS）分析重组α毒素的研究报道。在本研究中，我们报告了产气荚膜梭菌α毒素的成功克隆、表达、分泌、鉴定和序列测定。
