通过聚合酶链反应（PCR）扩增并使用表达载体克隆和表达艰难梭菌毒素A基因（tcdA）

Cloning and expression of Clostridium difficile toxin A gene (tcdA) by PCR amplification and use of an expression vector.

作者信息

Ackermann G, Löffler B, Tang-Feldman Y J, Cohen S H, Silva J, Rodloff A C

机构信息

Institute for Medical Microbiology and Epidemiology of Infectious Diseases, University of Leipzig, Liebigstrasse 24, 04103 Leipzig, Germany.

出版信息

Mol Cell Probes. 2004 Aug;18(4):271-4. doi: 10.1016/j.mcp.2004.02.002.

DOI:10.1016/j.mcp.2004.02.002

PMID:15271388

Abstract

Toxigenic Clostridium difficile isolates harbor a 19 kb pathogenicity locus that encodes the genes for toxins A and B. Toxins A and B are among the largest known bacterial toxins expressing potent cytotoxicity and enterotoxicity, and thus the major virulence factors in C. difficile associated diarrhea. Cloning and sequencing of toxin genes is of interest for studies of molecular pathogenesis. We report the amplification and cloning of the complete toxin A gene into an Escherichia coli expression vector. Ten clones analyzed contained the complete toxin A gene. Four of these clones showed cytotoxic activity in cell culture, and were positive for toxin A as determined by ELISA. Toxin A expression was confirmed by Western immunoblot analysis. The presence of cytotoxic activity in cell culture suggests that toxin A activity is independent of other genes in the pathogenicity locus.

摘要

产毒艰难梭菌分离株含有一个19 kb的致病位点，该位点编码毒素A和毒素B的基因。毒素A和毒素B是已知最大的细菌毒素之一，具有强大的细胞毒性和肠毒性，因此是艰难梭菌相关性腹泻的主要毒力因子。毒素基因的克隆和测序对于分子发病机制的研究具有重要意义。我们报告了将完整的毒素A基因扩增并克隆到大肠杆菌表达载体中。分析的10个克隆均含有完整的毒素A基因。其中4个克隆在细胞培养中表现出细胞毒性活性，通过ELISA检测确定为毒素A阳性。通过Western免疫印迹分析证实了毒素A的表达。细胞培养中细胞毒性活性的存在表明毒素A的活性独立于致病位点中的其他基因。