蕲蛇蛇毒中一种新型自降解抗凝血酶AA-ACA-I的纯化和表征

利用嗜硫色谱和POROS HQ20离子交换色谱从蕲蛇蛇毒中纯化得到一种新型P-Ⅲ型蛇毒金属蛋白酶（snake venom metalloproteases SVMP) AA-ACA-I.该酶经SDS-PAGE测得分子量为 47.6 kD,含有链内二硫键,加DTT处理后,分子量为50.8 kD,中性糖含量为3.5%,具有出血毒性和抗凝血活性.在70℃和pH 9.0条件下保温60 min,该酶会自降解成分子量30 kD左右的新蛋白,降解得到的新蛋白抗凝血活性高于原蛋白     

Sperm aggregations in female Agkistrodon piscivorus (Reptilia:Squamata): a histological and ultrastructural investigation

Upon copulation in female Agkistrodon piscivorus, sperm migrate up the oviduct to sperm storage tubules (SSTs) in the posterior infundibulum. The epithelium of the SSTs is composed of ciliated and secretory cells and differs ultrastructurally from that of the epithelium lining the lumen of the posterior infundibulum. Sperm pass through an area composed primarily of ciliated cells at the opening of each gland before aligning themselves in parallel arrays with their nuclei facing an area composed primarily of secretory cells at the base of the tubules. Sperm are also found embedded inter- and intracellularly in the SSTs. The secretory vacuoles in the SSTs become highly electron dense after the start of the fall mating season along with the synthesis of lipid droplets. Histochemical analysis reveals that the alteration in secretory material density is caused by the production of neutral carbohydrates. Some sperm remain in aggregates in the nonglandular section of the posterior uterus until the time of ovulation. However, ultrastructural evidence indicates these sperm degrade before ovulation. Therefore, sperm in posterior aggregates have no role in fertilization of ovulated ova. The data presented here support the hypothesis that infundibular sperm storage is the mode that snakes utilize to sequester viable sperm until ovulation.     

Renal sexual segment of the Cottonmouth snake, Agkistrodon piscivorous (Reptilia, Squamata, Viperidae)

The seasonal variation of the renal sexual segment (RSS) of males of the Cottonmouth snake, Agkistrodon piscivorous, is described using light and electron microscopy. This study is the first to describe the ultrastructure of the RSS of a viper (Viperidae) and only the fourth on a snake. Renal sexual segments from males collected February to May and from August to November are similar in appearance. The cells are eosinophilic and react with periodic acid/Schiff procedure (PAS) for neutral carbohydrates and bromphenol blue (BB) for proteins. At the ultrastructure level, the cells contain large (2 microm diameter), electron-dense secretory granules and smaller vesicles with a diffuse material, and these structures abut against the luminal border and upon clear vacuoles continuous with intercellular canaliculi. Evidence was found for both apocrine and merocrine processes of product release. In June and July, the RSS are significantly smaller in diameter, largely basophilic, and have only scattered granules that are PAS+ and BB+. Cytologically, the RSS from June to July lack electron-dense secretory granules and the smaller vesicles with diffuse material. Numerous condensing vacuoles and abundant rough endoplasmic reticulum, however, indicate that active product synthesis is occurring. This is the first report of significant seasonal variation in the histology and ultrastructure of the RSS of a snake, although such reports exist for lizards. The seasons when the RSS is most highly hypertrophied correspond to the fall and spring mating seasons of A. piscivorous, as determined by other studies.     

尖吻蝮蛇未知C类凝集素蛋白cDNA扩增、克隆与表达

蛇毒中C类凝集素(C-TLs)超家族蛋白,是具有抗凝血等功能的一族蛋白质.根据测得的蛇毒蛋白的N端氨基酸序列,设计PCR引物;从湖南尖吻蝮蛇毒腺中提取总RNA,经反转录,再通过温度降落锚式PCR,在只使用一个特异性引物和一个通用引物、进行一次循环数不超过30的非巢式反应的条件下,即获得两个较为清晰的扩增条带;其中之一带经克隆、测序、比较,证明其与已知的蛇毒中C类凝集素超家族蛋白质有较高的同源性.在大肠杆菌中获得高效融合表达,融合蛋白占细胞总蛋白的26%～30%.     

长白蝮蛇类凝血酶基因的克隆及分析

从长白蝮蛇（Agkistrodon halys Ussurin)毒腺中抽提总RNA,采用RT－PCR扩增其类凝血酶基因,经全序列测定,获得2个类凝血酶基因,ussurin和ussurase,它们全长分别为708和699个核苷酸,即分别编码236和233个氨基酸;根据同源性,推测它们的活性中心分别为His^43,Asp^88和Ser^182与His^40,Asp^85和Ser^179;二硫键分别为Cys^7-Cys^141,Cys^28-Cys^44,Cys^76-Cys^234,Cys^120-Cys^188,Cys^152-Cys^167和Cys^178-Cys^203;与Cys^7-Cys^138,Cys^25-Cys^41,Cys^73-Cys^231,Cys^117-Cys^185,Cys^149-Cys^164和Cys^175-Cys^200。该蛇毒类凝血酶cDNA序列及推导的氨基酸序列为首次报道。     

尖吻蝮蛇毒内一种新抗凝血因子ACF II 的纯化及特性

利用阴离子交换层析、凝胶过滤及阳离子交换层析三步方法, 从皖南尖吻蝮蛇毒中分离纯化到一个新的抗凝血因子ＡＣＦＩＩ（ａｎｔｉｃｏａｇｕｌａｔｉｏｎ ｆａｃｔｏｒＩＩ） 。纯化的ＡＣＦＩＩ在ＰＡＧＥ、ＳＤＳＰＡＧＥ和ＩＥＦＰＡＧＥ图谱上均呈单一区带。ＡＣＦＩＩ由两条分子量为１４．６ ｋＤ 的肽链通过二硫键连接在一起, 其等电点为７．０。ＡＣＦＩＩ具有显著的抗凝血活性, 在体外延长ＰＰＴ 时间的最终浓度为０ ．４ ｍｇ／Ｌ。ＡＣＦＩＩ不具有类凝血酶活性、磷脂酶Ａ２ 活性和纤溶活性,也没有出血活性和毒性, 是一种潜在的高效抗凝药物。     

尖吻蝮蛇毒磷脂酶A2基因的克隆与序列分析

从尖吻蝮蛇毒腺中抽提总ＲＮＡ,经ＲＴＰＣＲ扩增磷脂酶Ａ２的基因,以江浙蝮蛇的酸性磷脂酶Ａ２基因为探针杂交筛选克隆,分离得到４种磷脂酶Ａ２基因。经双向测序测定了这些磷脂酶Ａ２同功酶基因的全序列,并由此推导出编码的氨基酸序列。运用计算机软件推算了它们的等电点,按照等电点和结构特征将它们分别命名为尖吻蝮蛇毒酸性磷脂酶Ａ２Ｉ（Ａ．ａＡＰＬＡ２Ｉ）、尖吻蝮蛇毒酸性磷脂酶Ａ２ＩＩ（Ａ．ａＡＰＬＡ２ＩＩ）、尖吻蝮蛇毒碱性磷脂酶Ａ２（Ａ．ａＢＰＬＡ２）和尖吻蝮蛇毒Ｌｙｓ４９磷脂酶Ａ２（Ａ．ａＬｙｓ４９ＰＬＡ２）。其中Ａ．ａＡＰＬＡ２Ｉ的１～１０位氨基酸残基序列同以前分离得到的尖吻蝮蛇酸性磷脂酶Ａ２已测定的１～１０位氨基酸残基序列完全一致。Ａ．ａＬｙｓ４９ＰＬＡ２基因则由于其推导出的４９位氨基酸残基由Ｌｙｓ代替了Ａｓｐ而区别于以前克隆到的磷脂酶Ａ２基因。最后运用计算机软件比较了它们的同源性。这一组磷脂酶Ａ２基因的克隆,将为进一步研究磷脂酶Ａ２的结构与功能的关系提供更多的信息。     

蕲蛇酶在健康人的药物代谢动力学

健康自愿者１４人,男９人,女５人,除研究者本身（女,６４岁）外,平均年龄２５．８±ｓ６．５岁,分３组。分别一次ｉｖ蕲蛇酶注射液１Ｕ（１５０）,２Ｕ（３００）和３Ｕ（４５０μｇ）。用ＥＬＩＳＡ方法检测药后不同时间血药浓度以研究药代动力学。结果显示血药－时间曲线符合二室开放模型,其ｔ１／２α分别为０．６６±０．１０,０．９７±０．３４和１．２２±０．７７ｈ,ｔ１／２β分别为１５．９５±２．４,１８．８０±４．９４和１９．７３±４．２８ｈ     

浙江产蝮蛇Agkistrodon halys(Pallas)蛇毒磷酸二酯酶的酶学性质及其对大分子核酸的作用

1.以双对硝基苯磷酸钠为底物,测得蝮蛇毒磷酸二酯酶的最适pH在10左右,最适温度在60℃左右;酶在37℃和60℃的Km分别为5.65×10~(-4)M和7.97×10~(-4)M。 2.酶在pH5—10.5范围和50℃以下稳定。 3.钙离子和镁离子对酶活力有激活作用,但钡离子、镁离子、锌离子、二价铅离子、钴离子、二价和三价铁离子、锰离子、镍离子等都有不同程度地抑制作用。 4.除乙酸根离子外,碳酸根,硫酸根,乙二胺四乙酸根、磷酸根,钼酸根,酒石酸根,柠檬酸根,亚硝酸根,巴比妥酸根和硫代硫酸根等阴离子都有不同程度地抑制作用。 5.蝮蛇毒磷酸二酯酶能充分水解RNA和DNA但后者的水解速度比前者快许多。     

江浙蝮蛇磷脂酶A2基因的多样性研究

我们利用简并引物从江浙蝮蛇腺总ＲＮＡ经ＲＰ－ＰＣＲ扩增磷脂酶Ａ２（简称ＰＬＡ２）基因,并以碱性ＰＬＡ２（Ｂ－ＰＬＡ２）基因为探针,分离出了酸性ＰＬＡ２（Ａ－ＰＬＡ２）和两个未见报道的特征结构类同的基因,分别命名为Ａｓｎ＾４８－ＰＡＬ２和ＢＡ－ＰＡＬ２。双向测序测定了这组ＰＬＡ２同工酶（除信号肽外）基因的全序列,并由此推导编码的氨基酸序列。其中Ａ－ＰＬＡ２基因编码的氨基酸序列与较早报道的由蛇毒中分离