CLONING, NUCLEOTIDE SEQUENCING, AND EXPRESSION OF THE CLOSTRIDIUM-PERFRINGENS ENTEROTOXIN GENE IN ESCHERICHIA-COLI
CLONING, NUCLEOTIDE SEQUENCING, AND EXPRESSION OF THE CLOSTRIDIUM-PERFRINGENS ENTEROTOXIN GENE IN ESCHERICHIA-COLI
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DOI：
10.1128/iai.61.8.3429-3439.1993
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发表时间：
1993-08-01
影响因子：
3.1
通讯作者：
MCCLANE, BA
中科院分区：
文献类型：
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作者：
CZECZULIN, JR;HANNA, PC;MCCLANE, BA
产气荚膜梭菌肠毒素(CPE)是引起产气荚膜梭菌食物中毒和其他胃肠道疾病的重要毒力因子，其编码基因(CPE)已被克隆、测序并在大肠杆菌中表达。CPE基因编码一个319个氨基酸的多肽，推测其相对分子质量为35,317。在CPE的5‘端没有一个典型的信号肽的一致序列。定量免疫印迹分析表明，重组CPE阳性大肠杆菌的细胞裂解物中含有适量的CPE，对哺乳动物Vero细胞的细胞毒作用与天然CPE相当。CPE在重组大肠杆菌中的表达似乎主要由梭状芽胞杆菌启动子驱动。免疫印迹分析还表明，肠毒素阳性产气荚膜梭菌的营养细胞裂解物中的CPE水平很低。然而，当相同的产气荚膜梭菌菌株被诱导成孢子时，在这些产孢子细胞中检测到的CPE表达要比营养体产气假单胞菌或重组大肠杆菌强得多。综上所述，这些结果强烈表明，产孢子对CPE的表达不是必需的，但产孢量确实促进了CPE的高水平表达。
A complete copy of the gene (cpe) encoding Clostridium perfringens enterotoxin (CPE), an important virulence factor involved in C. perfringens food poisoning and other gastrointestinal illnesses, has been cloned, sequenced, and expressed in Escherichia coli. The cpe gene was shown to encode a 319-amino-acid polypeptide with a deduced molecular weight of 35,317. There was no consensus sequence for a typical signal peptide present in the 5' region of cpe. Cell lysates from recombinant cpe-positive E. coli were shown by quantitative immunoblot analysis to contain moderate amounts of CPE, and this recombinant CPE was equal to native CPE in cytotoxicity for mammalian Vero cells. CPE expression in recombinant E. coli appeared to be largely driven from a clostridial promoter. Immunoblot analysis also demonstrated very low levels of CPE in vegetative cell lysates of enterotoxin-positive C. perfringens. However, when the same C. perfringens strain was induced to sporulate, much stronger CPE expression was detected in these sporulating cells than in either vegetative C. perfringens cells or recombinant E. coli. Collectively, these results strongly suggest that sporulation is not essential for cpe expression, but sporulation does facilitate high-level cpe expression.