Quantitative detection of gene expression and toxin complex produced by Clostridium botulinum serotype D strain 4947
Quantitative detection of gene expression and toxin complex produced by Clostridium botulinum serotype D strain 4947
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DOI：
10.1016/j.mimet.2006.04.013
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发表时间：
2006-12-01
影响因子：
2.2
通讯作者：
Ohyama, Tohru
中科院分区：
文献类型：
--
作者：
Kouguchi, Hirokazu;Suzuki, Tomonori;Ohyama, Tohru
肉毒毒素是由肉毒梭菌产生的一种由神经毒素(NT)、无毒非血凝素(NTNHA)和血凝素亚组分(HA-70、HA-33和HA-17)非共价组装而成的大毒素复合体(L-TC)。本研究采用定量逆转录聚合酶链式反应技术，检测了肉毒杆菌D型菌株D-4947(D-4947)在细胞生长过程中5种L-TC组分的基因表达。在指数生长期中期(6.5h)检测到5个组分基因的转录，在生长稳定期早期(12h)达到最大值。NT和ntnHA的mRNA转录产物的比例约为1：1，表明NT和ntnHA是双顺反子转录的。另一方面，尽管HA-33基因的转录水平低于其他两个HA亚组分基因，但HA基因的转录水平却是NT和ntnHA基因的数倍。定量逆转录聚合酶链式反应的结果表明，肉毒杆菌TC相关蛋白中HA-33的缺失可以解释D-4947产生其他较小尺寸的L-TC(610、540和410 kDa)的原因，HA-33的分子量比成熟的650 kDa L-TC少。Western印迹分析表明，细胞裂解物中的TC最初出现在指数期中期，而胞外TC随后出现在稳定期的早期。(C)2006爱思唯尔B.V.保留所有权利。
Botulinum toxin is produced by Clostridium botulinum as a large toxin complex (L-TC) non-covalently assembled with a neurotoxin (NT), a non-toxic non-hemagglutinin (NTNHA) and hemagglutinin subcomponents (HA-70, HA-33, and HA-17). In this study, the gene expressions of five individual L-TC components were examined by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) in C. botulinum serotype D strain 4947 (D-4947) during cell growth. Transcripts for the five component genes were successfully detected in the mid-exponential growth phase (6.5 h), reaching a maximum at the early stationary growth phase (12 h). The ratio of the mRNA transcripts of nt and ntnha was approximately 1: 1, suggesting that nt and ntnha are bicistronically transcribed. On the other hand, the transcript levels of the ha genes were several-fold higher than those of nt and ntnha, although the mRNA transcript level of ha-33 was less than the other two ha subcomponent genes. The results based on qRT-PCR indicate that a shortage of HA-33 among the proteins associated with botulinum TC could explain the production by D-4947 of other smaller-sized L-TCs (610, 540 and 410 kDa) with fewer HA-33 molecules than the mature 650 kDa L-TC. Western blot analysis demonstrated that TC species in cell lysate were initially observed in the mid-exponential phase, while extracellular TCs were detected subsequently in the early stationary phase. (c) 2006 Elsevier B.V. All rights reserved.