蝮蛇类凝血酶基因的分析及表达研究

从蝮蛇（ＡｇｋｉｓｔｒｏｄｏｎｈａｌｙｓＰａｌａｓ）毒腺中抽提总ＲＮＡ,经ＲＴＰＣＲ扩增该基因,克隆后经全序列测定,蝮蛇类凝血酶ｐａｌａｓｅ的ｃＤＮＡ长７０８个核苷酸,即编码２３６个氨基酸;根据同源性,推测该类凝血酶ｐａｌａｓｅ的活性中心为Ｈｉｓ４１、Ａｓｐ８６和Ｓｅｒ１８２;二硫键为Ｃｙｓ７Ｃｙｓ１３９、Ｃｙｓ２６Ｃｙｓ４２、Ｃｙｓ７４Ｃｙｓ２３４、Ｃｙｓ１１８Ｃｙｓ１８８、Ｃｙｓ１５０Ｃｙｓ１６７和Ｃｙｓ１７８Ｃｙｓ２０３;该蝮蛇毒类凝血酶ｃＤＮＡ序列及推导的氨基酸序列均为首次报道。构建Ｔ７启动子控制下的ｐａｌａｓｅ的大肠杆菌表达质粒,ＩＰＴＧ诱导ｐａｌａｓｅ获得表达。     

尖吻蝮蛇毒内一种新的抗血小板凝集蛋白agkisacutacin的纯化及其性质

从皖南尖吻蝮蛇毒中经阴离子交换层析和凝胶过滤层析分离纯化得到抗血小板凝集蛋白agkisacutacin ,纯化的agkisacutacin由分子量为 1 4kD和 1 5kD的 2条肽链通过二硫键连接 ,能有效抑制ristocetin诱导的血小板凝集 (IC5 0 为 1 8.5mg/L) ,能轻微抑制凝血酶诱导的血小板聚集 (IC5 0 为 1 .2 2 g/L) ,但对ADP、胶原诱导的血小板聚集无影响。agkisacutacin不具有纤溶活性、抗凝活性、磷脂酶A2 活性和出血活性 ,是一种潜在的安全、有效的抗血小板性栓塞的药物     

江浙蝮蛇神经生长因子在大肠杆菌中的表达及纯化

将江浙蝮蛇（ＡｇｋｉｓｔｒｏｄｏｎｈａｌｙｓＰａｌａｓ,Ａ．ｈ．Ｐ．）神经生长因子（Ｎｅｒｖｅｇｒｏｗｔｈｆａｃｔｏｒ,ＮＧＦ）基因克隆于分泌型原核表达载体ｐＥＴ２２ｂ＋,以Ｃ端融合了６个组氨酸的形式在大肠杆菌ＢＬ２１（ＤＥ３）中进行了ＩＰＴＧ诱导表达。ＳＤＳＰＡＧＥ分析确定有一比理论值略大的诱导表达条带,其表达量占全菌蛋白质的２０％左右,且表达蛋白质主要是以包涵体的形式存在。用６ｍｏｌ／Ｌ的盐酸胍溶解包涵体后,利用固定化金属离子（Ｎｉ２＋）配体亲和层析一步纯化目的蛋白质,纯度可达８０％左右。对该重组肽进行变复性研究。利用ＰＣ１２细胞进行生物活力测定,证明表达产物具有ＮＧＦ生物活力。     

Purification and characterization of AHPM,a novel non-hemorrhagic P-IIIc metalloproteinase with α-fibrinogenolytic and platelet aggregation-inhibition activities,from Agkistrodon halys pallas venom

A novel non-hemorrhagic metalloproteinase, AHPM, was purified from the venom of Agkistrodon halys pallas by a combination of ion-exchange and gel filtration chromatography. AHPM is a dimeric glycoprotein with multiple pIs around pH 7.9 and has a molecular mass of 110 kDa with two blocked N-terminuses. Partial sequence of AHPM obtained by LC-MS/MS analysis together with its dimeric nature reveals that it is a P-IIIc snake venom metalloproteinase composed of metalloproteinase, disintegrin-like and cysteine-rich domains. AHPM has a conserved DECD sequence in the disintegrin-like domain. AHPM hydrolyzes casein and fibrinogen and also dissolves fibrin clots and the proteolytic activity is abolished by EDTA, but not by PMSF, suggesting that it is a metalloproteinase. The protease hydrolyzes rapidly the Aα-chain of fibrinogen followed by the Bβ-chain and does not cleave the γ-chain. AHPM contains endogenous Zn²⁺ and Ca²⁺ ions at a molar ratio of 1:1.9 and 1:4.2, respectively, and Zn²⁺ ions are essential for its proteolytic activity. AHPM inhibits collagen-and ADP-induced platelet aggregation with half maximal inhibitory concentrations of 200 ± 8 nM and 280 ± 10 nM, respectively. EDTA markedly attenuates the inhibition of ADP-induced platelet aggregation by AHPM, indicating that the fibrinogenolytic activity of AHPM is involved in its inhibition of ADP-induced platelet aggregation. AHPM is devoid of hemorrhagic activity when injected (up to 30 μg) subcutaneously into mice. AHPM is so far identified as first non-hemorrhagic P-IIIc SVMP which has both fibrinolytic and platelet aggregation-inhibition activities. The bifunctional enzyme may have a potential clinical application as a thrombolytic agent.     

中西医结合治疗蝮蛇咬伤致心功能损害与衰竭79例临床分析

本文报道了中西医结合治疗蝮蛇咬伤致心功能损害与衰竭79例的临床总结.笔者认为坚持解毒与排毒相结合,是保护、改善心功能的根本措施.按心脏及其他脏器功能损害情况,采用益气生脉、解毒护心等法,辨证施治,配合对症支持疗法,及调节水电解质与酸碱平衡等.保持内环境稳定,防治继发感染,是促进心功能恢复的有效方法。     

尖吻蝮蛇伤患者微循环与凝血象的同步观察及其与临床关系

本文对尖吻蝮蛇伤患者的甲皱微循环和凝血象同时进行了动态观察。结果显示尖吻蝮蛇伤患者中毒程度不同,甲皱微循环也发生不同程度的改变,另中毒程度越严重,凝血象变化也越显著.甲皱微循环和凝血象的变化与临床病情变化是一致的,对病情的转归及预后估计均有一定的指导意义。     

酶标法观察尖吻蝮出血毒素Ⅰ对毛细血管的作用

从尖吻蝮蛇毒中分离得到的出血毒素Ⅰ(AaHI)具有破坏血管系统并造成广泛出血的作用。本文报道用辣根过氧化物酶标记的AaHI可以特异地结合在毛细血管内皮细胞外膜上,由此推测内皮细胞膜上存在HaHI的受体或作用位点。     

江浙蝮蛇(Agkistrodon halys Pallas)毒的抑菌作用及其抑菌组分L-氨基酸氧化酶的分离纯化

我们用平皿测活法研究了江浙蝮蛇毒对真菌和大肠杆菌生长的抑制作用，并对其抗细菌的组分进行柱层析分离纯化，得到组分单一的LAO不仅有抑菌作用还有L-氨基酸氧化酶的活性。纯化后LAO的比活力为808U／mg。蛇毒经酸性聚丙烯酰胺电泳后测抑菌活性发现有3个抑菌组分(Ⅰ、Ⅱ、Ⅲ)，LAO为抑菌组分Ⅱ。