江浙蝮蛇蛇毒蛋白C激活因子基因的克隆及测序

从江浙蝮蛇毒腺中抽提总RNA，RT－PCR进行体外扩增，获得江浙蝮蛇蛇毒蛋白C激活因子基因，克隆至pGEX-5X-3载体中，对3个重组克隆分别作DNA全序列分析，通过遗传密码推导出相应的氨基酸序列，与其它已知的丝氨酸复白酶蛇毒蛋白的氨基酸作比较，其中许多上氨基酸有很强的同源性，该基因的成功克隆，不仅推导出江浙蝮蛇蛇毒蛋白C激活因子的蛋白质序列，也为进一步开展江浙蝮蛇蛇毒蛋白C激活因子蛋白质工程的研究工作打下良好的基础。     

Calcium Ion-Induced Stabilization and Refolding of Agkisacutacin from <Emphasis Type="Italic">Agkistrodon Acutus</Emphasis> Venom Studied by Fluorescent Spectroscopy

Agkisacutacin isolated from the venom of Agkistrodon acutus is a coagulation factor IX / coagulation factor X-binding protein with marked anticoagulant- and platelet-modulating activities. Ca²⁺ ion-induced stabilization and refolding of Agkisacutacin have been studied by following fluorescent measurements. Ca²⁺ ions not only increase the structural stability of agkisacutacin against GdnHCl denaturation, but also induce its refolding. The GdnHCl-induced unfolding of the apo-agkisacutacin and the purified agkisacutacin is a single-step process with no detectable intermediate state. Ca²⁺ ions play an important role in the stabilization of the structure of agkisacutacin. Ca²⁺-stabilized agkisacutacin exhibits higher resistance to GdnHCl denaturation than the apo-agkisacutacin. It is possible to induce refolding of the unfolded apo-agkisacutacin merely by adding 1 mM Ca²⁺ ions without changing the concentration of the denaturant. The kinetic result of Ca²⁺-induced refolding provides evidences for that agkisacutacin consists of at least two refolding phases and the first phase of Ca²⁺-induced refolding should involve the formation of the compact Ca²⁺-binding site regions, and subsequently, the protein undergoes further conformational rearrangements to form the native structure.     

高效离子交换色谱法分离皖南尖吻蝮蛇毒活性组分

采用高效离子交换色谱法对经初步分离的皖南尖吻蝮蛇毒活性成分进行进一步的分离纯化。采用线性梯度洗脱,得到蝮蛇毒活性组分纯品,并用峰面积归一化法计算其含量为４５．４１％左右,纯度在９０％以上。     

Preparation of a Monoclonal Antibody against a Kallikrein-Like Enzyme from Agkistrodon halys pallas Venom and Its Application in a Pharmacokinetic Study

Snake venom contains bioactive materials for drug development, diagnosis, and treatment. After separating and purifying the kallikrein-like enzyme (AHP-Ka) from Agkistrodon halys pallas venom for the first time, a monoclonal antibody against AHP-Ka was prepared and characterized. An indirect sandwich enzyme-linked immunosorbent assay (ELISA) based on the monoclonal antibody was developed and validated for the pharmacokinetic analysis of AHP-Ka in rat plasma. The method was calibrated using rat plasma and 1:100 dilution of plasma was selected to prepare a calibration curve to validate the precision, accuracy, and stability of the ELISA method. A good linear relationship was obtained in a working range from 3.9 ng/mL to 62.5 ng/mL with a limit of detection of 2.94 ng/mL. Intra- and inter-batch precision were less than 10%. The average recovery ranged from 94.6% to 104.4% in rat plasma at the concentrations of 5 ng/mL, 15 ng/mL, and 45 ng/mL, respectively. The ELISA method was successfully used for the pharmacokinetic study of AHP-Ka in Sprague-Dawley rat plasma after intravenous administration. The work is expected to contribute to future preclinical development of AHP-Ka.     

三步柱色谱法从江浙蝮蛇蛇毒中分离纯化类凝血酶

建立了一种用CM Sepharose CL-6B阳离子交换、DEAE Sepharose Fast Flow阴离子交换和Sephadex G-75凝胶排阻三步柱色谱从江浙蝮蛇蛇毒中分离纯化类凝血酶的方法。在实验室小柱分离方案的基础上,对该纯化工艺进行了放大。当上样量达实验室小柱的25倍时,所得类凝血酶的质量指标与实验室小柱基本一致。采用该法所得的蝮蛇类凝血酶经Shim-pack Diol-300高效凝胶排阻柱测得其相对分子质量约为33500,用Shim-pack VP-ODS反相色谱柱检测其纯度约为96%。从江浙粗蛇毒中提取类凝血酶时,类凝血酶的总质量收率约为0.3%,总活性收率约为64%,比活可达2000 U/mg。     

毛细管区带电泳分析五步蛇毒蛋白C激活剂和蛋白C的相互作用

建立了毛细管区带电泳分析五步蛇毒蛋白C激活剂(protein C activator, PCA)与血浆蛋白C(protein C, PC)的相互作用。以未涂层毛细管(60.2 cm(有效长度50 cm)×75 μm)为分离柱,50 mmol/L Tris-HCl(pH 7.4)为运行缓冲液,于198 nm波长下检测。对影响五步蛇毒PCA分离的因素(如缓冲液、离子浓度)及在37.5 ℃下孵育不同时间的五步蛇毒PCA与PC的相互作用进行考察。方法的检出限(以信噪比为3计)为3 mg/L,线性范围为10～300 mg/L。五步蛇毒PCA迁移时间和峰面积的相对标准偏差分别为0.56%和3.8%(n=6)。等体积的五步蛇毒PCA(200 mg/L)与PC(60 mg/L)孵育5 min,其结合率达到最大,且谱图中未见有PC水解的肽链。该五步蛇毒PCA可改变PC空间构象直接激活PC。该方法简单,灵敏度和分辨率高,分析结果为今后快速检测五步蛇毒PCA及其活性提供了重要的理论依据。     

正交晶型江浙蝮蛇毒碱性磷脂酶A2初步结晶学研究

The basic phospholipase A2 from the venom of Agkistrodon halys pallas possesses the ability to cause hemolysis in contrast to the other two phospholipase A2 from the same venom. A new form of crystals of this enzyme was grown. The crystals belong to space group of P212121 with unit cell parameters of a=9.175nm, b=10.080nm , c=2.287nm.The crystals diffract to high resolution, and are suitable for detailed structural studies. The data were collected up to 0.25nm resolution using synchrotron radiation-imaging plate-Weissenberg camera system. Preliminary analysis reveals the presence of two molecules in the asymmetric uint and the molecule may pack with the smallest dimension approximately parallel to the c axis. The new crystal form is more attractive than the monoclinic one previously reported for crystallographic structure determination as it contains fewer molecules in the asymmetric unit.     

长白山白眉蝮蛇蛇毒精氨酸酯酶1的研究Ⅰ.纯化、分子量测定及纯度鉴定

利用离子交换 (DEAESephadexA - 50 )和凝胶过滤层析 (SephadexG - 75)技术首次从长白山白眉蝮蛇蛇毒 (AgkistrodonblomhoffiiUssurensis)中纯化得到了一种精氨酸酯酶纯品。经SDS -聚丙烯酰胺凝胶电泳(SDS -PAGE)以及基质辅助激光解吸电离飞行时间质谱 (MALDI/TOF/MS)鉴定为单一纯蛋白 ,分子量为2 991 8.5± 1 5Da ,为进一步研究其结构与功能提供了可靠的依据。     

白眉蝮蛇蛇毒和蛇毒酶的基质辅助激光解吸电离飞行时间质谱法研究

应用基质辅激光解吸电离飞行时间质谱（ＭＡＬＤＩ－ＴＯＦ－ＭＳ）法对长白山眉蝮蛇蛇毒和纯化得到的两种蛇毒酶进行了研究,得到了它们的分子质量并验证了纯度。同时还考察了不同产地的蛇毒、蛇毒蛋白浓度以及基质对分析结果的影响,实验结果表明ＭＡＬＤＩ－ＴＯＦ－ＭＳ法是检测蛋白纯化过程和分析蛋白相对分子质量十分有效的手段。