湖南永州尖吻蝮蛇蛇毒的蛋白组分及毒性分析

Characterization of protein components and toxicity of Deinagkistrodon acutus venom in Yongzhou of Hunan

采用Nano–LC–ESI–MS/MS蛋白鉴定技术对湖南永州尖吻蝮蛇蛇毒蛋白组分进行质谱分析,应用常规体外试管法对尖吻蝮蛇蛇毒体外溶血值进行检测,采用改良寇式法对尖吻蝮蛇蛇毒LD50进行测定。结果显示:尖吻蝮蛇蛇毒含有50种蛋白,蛋白相对分子质量集中在1.0×104~2.0×104(46.9%)、4.0×104~5.0×104(22.4%)、2.0×104~3.0×104(10.6%)和7.0×104~8.0×104(7.6%),其中代表性的高丰度蛋白有蛇毒金属蛋白酶A(11.7%)、蛇毒金属蛋白酶H1(9.8%)、蕲蛇类凝血酶–2(7.3%)、抗凝血酶A–A亚基(6.8%),低丰度蛋白(<0.1%)有Ecto–5'–核苷酸酶、碱性磷脂酶A2 DAV–N6、生长分化因子11;蛇毒引起红细胞溶血的最低质量浓度为60.00μg/mL,尖吻蝮蛇蛇毒对KM小鼠的LD50为7.179 6 mg/kg,攻毒小鼠出现呼吸急促、躁动不安、注射部位出现奇特瘙痒和大面积溃烂,至死亡。

The protein components of the venom obtained from Deinagkistrodon acutus in Yongzhou area of Hunan were identified and characterized using Nano-LC-ESI-MS/MS.The in vitro hemolysis of D.acutus venom was conducted with use of the conventional test tube method,and the LD50 of the venom was measured using a challenge test in a KM mouse model.Fifty types of proteins were identified,and the protein relative molecular mass are mainly distributed in 1.0×104~2.0×104(46.9%),4.0×104~5.0×104(22.4%),2.0×104~3.0×104(10.6%)and 7.0×104~8.0×104(7.6%)containing high-abundance of representative proteins including snake venom metalloproteinase acutolysin-A(11.7%),snake venom metalloproteinase H1(9.8%),thrombin-like enzyme acutobin-2(7.3%),antithrombin A subunit A(6.8%)and low-abundance proteins(<0.1%)containing ecto-5'-nucleotidase,basic phospholipase A2 DAV-N6,growth/differentiation factor 11.The minimal erythrocyte hemolysis concentration and LD50 of D.acutus venom were 60.00μg/mL and 7.179 6 mg/kg,respectively.The typical clinical symptoms of challenged mice showed to be tachypnea,restlessness,unusual pruritus and widespread canker near the injection site and even to be repeatedly scratched,and death.