经钴-60γ辐射的破伤风毒素酶活性的表征与评估。
Characterization and evaluation of the enzymatic activity of tetanus toxin submitted to cobalt-60 gamma radiation.
作者信息
Sartori Giselle Pacifico, da Costa Andréa, Macarini Fernanda Lúcio Dos Santos, Mariano Douglas Oscar Ceolin, Pimenta Daniel Carvalho, Spencer Patrick Jack, Nali Luiz Henrique da Silva, Galisteo Andrés Jimenez
机构信息
Laboratory of Protozoology, Institute of Tropical Medicine, University of São Paulo (IMT/FMUSP), São Paulo, SP, Brazil.
Anaerobic Vaccines Section, Butantan Institute, São Paulo, SP, Brazil.
出版信息
J Venom Anim Toxins Incl Trop Dis. 2021 Apr 30;27:e20200140. doi: 10.1590/1678-9199-JVATITD-2020-0140.
BACKGROUND
Tetanus toxin blocks the release of the inhibitory neurotransmitters in the central nervous system and causes tetanus and its main form of prevention is through vaccination. The vaccine is produced by inactivation of tetanus toxin with formaldehyde, which may cause side effects. An alternative way is the use of ionizing radiation for inactivation of the toxin and also to improve the potential immunogenic response and to reduce the post-vaccination side effects. Therefore, the aim of this study was to characterize the tetanus toxin structure after different doses of ionizing radiation of Co.
METHODS
Irradiated and native tetanus toxin was characterized by SDS PAGE in reducing and non-reducing conditions and MALD-TOF. Enzymatic activity was measured by FRET substrate. Also, antigenic properties were assessed by ELISA and Western Blot data.
RESULTS
Characterization analysis revealed gradual modification on the tetanus toxin structure according to doses increase. Also, fragmentation and possible aggregations of the protein fragments were observed in higher doses. In the analysis of peptide preservation by enzymatic digestion and mass spectrometry, there was a slight modification in the identification up to the dose of 4 kGy. At subsequent doses, peptide identification was minimal. The analysis of the enzymatic activity by fluorescence showed 35 % attenuation in the activity even at higher doses. In the antigenic evaluation, anti-tetanus toxin antibodies were detected against the irradiated toxins at the different doses, with a gradual decrease as the dose increased, but remaining at satisfactory levels.
CONCLUSION
Ionizing radiation promoted structural changes in the tetanus toxin such as fragmentation and/or aggregation and attenuation of enzymatic activity as the dose increased, but antigenic recognition of the toxin remained at good levels indicating its possible use as an immunogen. However, studies of enzymatic activity of tetanus toxin irradiated with doses above 8 kGy should be further analyzed.
背景
破伤风毒素会阻断中枢神经系统中抑制性神经递质的释放，从而引发破伤风，其主要预防方式是通过接种疫苗。该疫苗是通过用甲醛使破伤风毒素失活来生产的，这可能会导致副作用。另一种方法是利用电离辐射使毒素失活，同时提高潜在的免疫原性反应并减少接种后的副作用。因此，本研究的目的是表征经不同剂量钴源电离辐射后的破伤风毒素结构。
方法
通过在还原和非还原条件下的SDS-PAGE以及基质辅助激光解吸电离飞行时间质谱（MALDI-TOF）对辐照后的破伤风毒素和天然破伤风毒素进行表征。通过荧光共振能量转移（FRET）底物测定酶活性。此外，通过酶联免疫吸附测定（ELISA）和蛋白质印迹数据评估抗原特性。
结果
表征分析显示，随着剂量增加，破伤风毒素结构逐渐发生改变。在较高剂量下还观察到了蛋白质片段的断裂和可能的聚集。在通过酶消化和质谱分析肽段保留情况时，在剂量达到4千戈瑞之前，鉴定结果仅有轻微改变。在后续剂量下，肽段鉴定结果极少。通过荧光分析酶活性表明，即使在较高剂量下，活性也衰减了35%。在抗原性评估中，检测到针对不同剂量辐照毒素的抗破伤风毒素抗体，随着剂量增加抗体逐渐减少，但仍保持在令人满意的水平。
结论
电离辐射促使破伤风毒素发生结构变化，如断裂和/或聚集，且随着剂量增加酶活性衰减，但毒素的抗原识别仍保持在良好水平，表明其有可能用作免疫原。然而，对于剂量高于8千戈瑞的辐照破伤风毒素的酶活性研究应进一步分析。
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