Lee Yu-Ching, Chen Wang-Chuan, Liang Meng-Huei, Lee Chi-Hsin, Tsai Keng-Chang, Chiang Jen-Ron, Chiang Liao-Chun, Chen Chi-Ching, Chang Chang-Yu, Lee Ching-Hsiao, Leu Sy-Jye, Yang Yi-Yuan
The Center of Translational Medicine, Taipei Medical University, Taipei, Taiwan; Antibody and Hybridoma Core Facility, Taipei Medical University, Taipei, Taiwan.
The School of Chinese Medicine for Post Baccalaureate, I-Shou University, Kaohsiung, Taiwan; Department of Chinese Medicine, E-Da Hospital, Kaohsiung, Taiwan.
Biochem Biophys Res Commun. 2015 May 1;460(2):170-6. doi: 10.1016/j.bbrc.2015.02.169. Epub 2015 Mar 10.
Trimeresurus mucrosquamatus (TM) is one of majorities of snake envenomation with necrotic and hemorrhagic toxin in Taiwan. In this study, chickens were used as an alternative animal model for immunization with TM venom. Using phage display technology to process four rounds of panning, selected single chain variable fragments (scFv) could specifically recognize TM venom proteins, which were later identified as a group of homogeneous venom serine protease. The specific scFv antibodies showed various inhibitory effects on sheep RBC lysis induced by TM venom using an indirect hemolytic assay in vitro. In addition, the survival times of mice were extended to certain degrees when treated with these scFv antibodies individually or in a combination. To elucidate the inhibitory mechanism, we used molecular modeling to build up the serine protease structure to simulate the possible interactions with scFv antibodies. The results suggested that the CDR-loop of the scFv antibodies (3S10 or 4S1) might bind at the 99-loop of venom serine protease so as to affect substrate access due to the partial collapse of the subsite S2 and the partial movement of the subsite S4. It is hoped these chicken-derived antibodies could be applied to develop diagnostic and therapeutic agents against snakebites.
台湾竹叶青蛇（TM）是台湾地区导致坏死性和出血性毒素中毒的主要蛇类之一。在本研究中，鸡被用作免疫TM毒液的替代动物模型。利用噬菌体展示技术进行四轮淘选，筛选出的单链可变片段（scFv）能够特异性识别TM毒液蛋白，随后鉴定为一组同源的毒液丝氨酸蛋白酶。在体外间接溶血试验中，特异性scFv抗体对TM毒液诱导的绵羊红细胞裂解表现出不同程度的抑制作用。此外，单独或联合使用这些scFv抗体治疗时，小鼠的存活时间有一定程度的延长。为阐明抑制机制，我们利用分子建模构建丝氨酸蛋白酶结构，以模拟其与scFv抗体的可能相互作用。结果表明，scFv抗体（3S10或4S1）的互补决定区环可能结合在毒液丝氨酸蛋白酶的99环处，由于亚位点S2的部分塌陷和亚位点S4的部分移动，从而影响底物的进入。希望这些鸡源抗体可用于开发抗蛇咬的诊断和治疗药物。
