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白唇竹叶青蛇毒和圆斑蝰蛇毒的体外免疫反应性及蛋白质分析比较

Comparative in vitro immunoreactivity and protein analysis of Trimeresurus albolabris and Tropidolaemus wagleri venoms.

作者信息

Reamtong Onrapak, Pearngam Phorutai, Laoungbua Panithi, Sitprija Siravit, Thiangtrongjit Tipparat, Srisuk Godchakorn, Vasaruchapong Taksa, Khow Orawan, Noiphrom Jureeporn, Chaiyabutr Narongsak, Chanhome Lawan, Kumkate Supeecha

机构信息

Department of Molecular Tropical Medicine and Genetics, Faculty of Tropical Medicine, Mahidol University, Ratchathewi, Bangkok, 10400, Thailand.

Science Division, International College, Mahidol University, Salaya, Phutthamonthon, Nakhon Pathom, 73170, Thailand.

出版信息

Sci Rep. 2025 Apr 12;15(1):12693. doi: 10.1038/s41598-025-97032-0.

DOI:10.1038/s41598-025-97032-0
PMID:40221593
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC11993613/
Abstract

Snakebite envenomation remains a significant global health concern, with antivenoms being the primary treatment. However, variations in venom composition can affect antivenom efficacy, leading to differences in immunoreactivity. This study aimed to evaluate and compare the immunological reactivity of venom components in Trimeresurus albolabris and Tropidolaemus wagleri venoms and further investigate the differences in antigenic properties of a key protein between two species that may influence antivenom recognition. The levels of immunological reactivity of monovalent (homospecific) antivenom and hemato polyvalent antivenom to Trimeresurus albolabris and Tropidolaemus wagleri venoms were evaluated using indirect ELISA. The immunoreactive levels of both antivenoms to antigenic proteins in Trimeresurus albolabris venom were comparable. In addition, both antivenoms reacted immunologically with antigens in Tropidolaemus wagleri venom. However, the hemato polyvalent antivenom showed greater reactivity to Tropidolaemus wagleri venom than the monovalent antivenom. The overall reactivity of the antivenoms to Trimeresurus albolabris venom was higher than that to Tropidolaemus wagleri venom. Using two-dimensional (2DE) immunoblotting and liquid chromatography mass-spectrometry-based proteomic technology (LC-MS/MS), immunoreactive and non-reactive proteins in both pit viper venoms were characterized and identified. Trimeresurus albolabris venom comprised a total of 235 spots, while Tropidolaemus wagleri venom contained 72 spots. Immunorecognition between the polyvalent antivenom and specific proteins in both venoms was mostly detected in proteins with a size over 30 kDa. Among the nine protein families identified in both venoms, the most frequently reactive proteins found in Trimeresurus albolabris venom were snake venom metalloproteinases (SVMP) and snake venom serine proteases (SVSP), while in Tropidolaemus wagleri venom, the most frequent were members of the L-amino acid oxidase (LAAO) family. For the non-immunoreactive proteins, we detected the highest identity numbers of phospholipase A (PLA) in Trimeresurus albolabris venom and SVSP in Tropidolaemus wagleri venom. The distinctive characteristics between the non-reactive SVSP in Tropidolaemus wagleri venom and the reactive SVSP in Trimeresurus albolabris venom were investigated. The antigenic properties and predicted B cell epitopes were further analyzed using a computational approach. Structural and physicochemical analyses indicated that Loop 2 (residues 100-110) in the immunoreactive SVSP from Trimeresurus albolabris venom exhibited higher hydrophilicity and surface accessibility compared to the non-immunoreactive SVSP from Tropidolaemus wagleri venom. These findings provide important insights into the differences in antivenom reactivity to specific proteins across different snake venoms and may contribute to future research aimed at optimizing antivenom formulations.

摘要

蛇咬伤中毒仍然是一个重大的全球健康问题，抗蛇毒血清是主要的治疗方法。然而，毒液成分的差异会影响抗蛇毒血清的疗效，导致免疫反应性的差异。本研究旨在评估和比较白唇竹叶青蛇毒和圆斑蝰蛇毒中毒液成分的免疫反应性，并进一步研究两种蛇毒中可能影响抗蛇毒血清识别的关键蛋白质的抗原特性差异。使用间接酶联免疫吸附测定法（ELISA）评估单价（同种特异性）抗蛇毒血清和血液多价抗蛇毒血清对白唇竹叶青蛇毒和圆斑蝰蛇毒的免疫反应水平。两种抗蛇毒血清对白唇竹叶青蛇毒中抗原性蛋白质的免疫反应水平相当。此外，两种抗蛇毒血清均与圆斑蝰蛇毒中的抗原发生免疫反应。然而，血液多价抗蛇毒血清对圆斑蝰蛇毒的反应性比单价抗蛇毒血清更高。抗蛇毒血清对白唇竹叶青蛇毒的总体反应性高于对圆斑蝰蛇毒的反应性。使用二维（2DE）免疫印迹和基于液相色谱-质谱联用的蛋白质组学技术（LC-MS/MS），对两种蝰蛇科蛇毒中的免疫反应性和非反应性蛋白质进行了表征和鉴定。白唇竹叶青蛇毒共包含235个斑点，而圆斑蝰蛇毒包含72个斑点。多价抗蛇毒血清与两种蛇毒中特定蛋白质之间的免疫识别大多在分子量超过30 kDa的蛋白质中检测到。在两种蛇毒中鉴定出的九个蛋白质家族中，白唇竹叶青蛇毒中最常发生反应的蛋白质是蛇毒金属蛋白酶（SVMP）和蛇毒丝氨酸蛋白酶（SVSP），而在圆斑蝰蛇毒中，最常见的是L-氨基酸氧化酶（LAAO）家族的成员。对于非免疫反应性蛋白质，我们在白唇竹叶青蛇毒中检测到磷脂酶A（PLA）的最高一致性编号，在圆斑蝰蛇毒中检测到SVSP的最高一致性编号。研究了圆斑蝰蛇毒中无反应性的SVSP与白唇竹叶青蛇毒中有反应性的SVSP之间的独特特征。使用计算方法进一步分析了抗原特性和预测的B细胞表位。结构和物理化学分析表明，与圆斑蝰蛇毒中无免疫反应性的SVSP相比，白唇竹叶青蛇毒中有免疫反应性的SVSP中的环2（第100-110位氨基酸残基）表现出更高的亲水性和表面可及性。这些发现为不同蛇毒对特定蛋白质的抗蛇毒血清反应性差异提供了重要见解，并可能有助于未来旨在优化抗蛇毒血清配方的研究。

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