Lee Chi-Hsin, Lee Yu-Ching, Liang Meng-Huei, Leu Sy-Jye, Lin Liang-Tzung, Chiang Jen-Ron, Yang Yi-Yuan
Graduate Institute of Medical Sciences, College of Medicine, Taipei Medical University, Taipei, Taiwan.
The Center of Translational Medicine, Taipei Medical University, Taipei, Taiwan Ph.D. Program for Biotechnology in Medicine, College of Medical Science and Technology, Taipei Medical University, Taipei, Taiwan Core Facility for Antibody Generation and Research, Taipei Medical University, Taipei, Taiwan.
Appl Environ Microbiol. 2015 Oct 16;82(1):71-80. doi: 10.1128/AEM.02608-15. Print 2016 Jan 1.
Snake venom protein from Deinagkistrodon acutus (DA protein), one of the major venomous species in Taiwan, causes hemorrhagic symptoms that can lead to death. Although horse-derived antivenin is a major treatment, relatively strong and detrimental side effects are seen occasionally. In our study, yolk immunoglobulin (IgY) was purified from eggs, and DA protein was recognized using Western blotting and an enzyme-linked immunosorbent assay (ELISA), similar to therapeutic horse antivenin. The ELISA also indicated that specific IgY antibodies were elicited after the fifth booster, plateaued, and lasted for at least 3 months. To generate monoclonal single-chain variable fragment (scFv) antibodies, we used phage display technology to construct two libraries with short or long linkers, containing 6.24 × 10(8) and 5.28 × 10(8) transformants, respectively. After four rounds of biopanning, the eluted phage titer increased, and the phage-based ELISA indicated that the specific clones were enriched. Nucleotide sequences of 30 individual clones expressing scFv were analyzed and classified into four groups that all specifically recognized the DA venom protein. Furthermore, based on mass spectrometry, the scFv-bound protein was deduced to be snake venom metalloproteinase proteins. Most importantly, both IgY and mixed scFv inhibited the lethal effect in mice injected with the minimum lethal dosage of the DA protein. We suggest that together, these antibodies could be applied to the development of diagnostic agents or treatments for snakebite envenomation in the future.
来自台湾主要毒蛇种类之一的尖吻蝮蛇毒蛋白（DA蛋白）会引发可导致死亡的出血症状。尽管马源抗蛇毒血清是主要的治疗方法，但偶尔会出现相对较强且有害的副作用。在我们的研究中，从鸡蛋中纯化出卵黄免疫球蛋白（IgY），并通过蛋白质印迹法和酶联免疫吸附测定（ELISA）来识别DA蛋白，其效果与治疗用马抗蛇毒血清类似。ELISA还表明，在第五次加强免疫后可诱导产生特异性IgY抗体，抗体水平达到稳定状态，并持续至少3个月。为了产生单克隆单链可变片段（scFv）抗体，我们利用噬菌体展示技术构建了两个分别带有短接头或长接头的文库，分别包含6.24×10⁸和5.28×10⁸个转化子。经过四轮生物淘选后，洗脱的噬菌体滴度增加，基于噬菌体的ELISA表明特异性克隆得到了富集。对30个表达scFv的单个克隆的核苷酸序列进行分析，并分为四组，所有组均能特异性识别DA蛇毒蛋白。此外。基于质谱分析，推断与scFv结合的蛋白为蛇毒金属蛋白酶蛋白。最重要的是，IgY和混合scFv均能抑制给小鼠注射最小致死剂量DA蛋白后的致死效应。我们认为，这些抗体未来可共同应用于蛇咬伤中毒的诊断试剂或治疗方法的开发。
