...
  • 主题
高级搜索  >
历史搜索 清空历史
首页> 外文期刊>Vaccine >Investigation of the detoxification mechanism of formaldehyde-treated tetanus toxin
【24h】

Investigation of the detoxification mechanism of formaldehyde-treated tetanus toxin

机译：甲醛处理的破伤风毒素的解毒机理研究

获取原文
获取原文并翻译 | 示例
           
页面导航
  • 摘要
  • 著录项
  • 相似文献
  • 相关主题

摘要

The tetanus vaccine is based on the extremely potent tetanus neurotoxin (TeNT), which is converted by treatment with formaldehyde and lysine into the non-toxic, but still immunogenic tetanus toxoid (TTd). This formaldehyde-induced detoxification, which to a large extend determines the quality and properties of the vaccine component, occurs through partly unknown chemical modifications of the toxin. The aim of this study was to gain knowledge of the detoxification mechanism in the generation of the tetanus vaccine. Two approaches were chosen: (i) the effect of changes in the concentrations of lysine and formaldehyde in the detoxification process and (ii) characterisation of the chemically detoxified TTd. (i) We examined a number of TTd components that was produced by varying the concentrations of formaldehyde and lysine during the inactivation. Toxicity tests showed that the detoxification failed when the lysine or formaldehyde concentration was < or =1/5 or < or =1/10, respectively, of the standard level. Gel-electrophoretic analyses showed that inter-chain cross-linking was formaldehyde-dependent and, furthermore, revealed that inter-chain cross-linking was not the only requirement for the inactivation. In addition, the measurable amount of tyrosine correlated inversely with the degree of inter-chain cross-linking. (ii) To study the formaldehyde-induced chemical modifications, the TTd was investigated using protein chemical techniques in combination with mass spectrometry (MS). Using off-line liquid chromatography (LC)-MS, the most pronounced chemical modifications were characterised as unstable Schiff-bases (+12 Da) located on lysine residues and the N-termini of peptides throughout the molecule. Several arginine residues were also found with +12 Da modifications due to Schiff-base formation or as a consequence of degenerative fragmentation of lysine/formaldehyde adducts or cross-links during MS. A few tyrosine residues were similarly observed with a mass increase of 12 Da. Even though it cannot be ruled out that this is a residual mass of higher molecule adducts or cross-links to tyrosine, amino acid analysis and MS data indicated that the modification forms a ring structure from a carbon in the aromatic ring to the backbone N(alpha). In addition, several mono-varepsilon-methyllysines (+14 Da) were observed as a likely consequence of reductive methylation of the Schiff-bases. A substantial part (87%) of the known TeNT sequence, including the active site, was covered using the off-line LC-MS approach to investigate the tryptic digested TTd. In contrast to the results obtained from the gel-electrophoretic experiments, neither intra/inter-chain cross-links nor cross-links to external lysines were observed in the MS analysis. Instability of the cross-links during separation and/or MS is likely to explain their absence in the analyses. The biological relevance of the observed modifications is discussed in relation to 3D mapping analyses. Proposals for the TeNT detoxification are discussed, although no direct evidence for the exact mechanism could be obtained.
机译：破伤风疫苗基于极强的破伤风神经毒素（TeNT），可通过甲醛和赖氨酸处理将其转化为无毒但仍具有免疫原性的破伤风类毒素（TTd）。这种甲醛引起的排毒，在很大程度上决定了疫苗成分的质量和特性，是通过部分未知的毒素化学修饰而发生的。这项研究的目的是了解破伤风疫苗生产中的解毒机理。选择了两种方法：（i）赖氨酸和甲醛浓度变化在解毒过程中的影响，以及（ii）化学解毒的TTd的表征。 （i）我们研究了通过在灭活过程中改变甲醛和赖氨酸的浓度而产生的许多TTd组分。毒性试验表明，当赖氨酸或甲醛的浓度分别为标准水平的<或= 1/5或<或= 1/10时，排毒失败。凝胶电泳分析表明链间交联是甲醛依赖性的，此外，链间交联不是灭活的唯一要求。此外，可测量的酪氨酸量与链间交联度成反比。 （ii）为了研究甲醛诱导的化学修饰，使用蛋白质化学技术结合质谱（MS）研究了TTd。使用离线液相色谱（LC）-MS，最明显的化学修饰的特征是位于整个分子中赖氨酸残基和肽N-末端的不稳定席夫碱（+12 Da）。由于席夫碱的形成或MS期间赖氨酸/甲醛加合物或交联的变性断裂，还发现了几个精氨酸残基，修饰度为+12 Da。类似地观察到一些酪氨酸残基，质量增加了12 Da。即使不能排除这是高分子量加合物或与酪氨酸交联的残留物，氨基酸分析和质谱数据表明该修饰形成了从芳环中的碳到主链N（ α）。此外，观察到几种单-半胱氨酸-甲基赖氨酸（+14 Da）可能是席夫碱还原性甲基化的结果。使用离线LC-MS方法研究了胰蛋白酶消化的TTd，覆盖了包括活性位点在内的大部分已知TeNT序列（87％）。与从凝胶电泳实验获得的结果相反，在质谱分析中既未观察到链内/链间交联，也未观察到与外部赖氨酸的交联。分离和/或MS期间交联的不稳定性很可能解释了分析中交联的缺失。观察到的修饰的生物学相关性将与3D映射分析进行讨论。讨论了有关TeNT排毒的建议，尽管无法获得确切机理的直接证据。

著录项

相似文献

  • 外文文献
  • 中文文献
  • 专利
获取原文

客服邮箱：kefu@zhangqiaokeyan.com

京公网安备：11010802029741号 ICP备案号：京ICP备15016152号-6 六维联合信息科技 (北京) 有限公司©版权所有

  • 客服微信

  • 服务号