蛇毒纤维蛋白（原）溶解酶的研究进展

蛇毒纤溶酶能直接溶解纤维蛋白（原）,具有作为强力溶栓剂的潜在价值。对蛇毒纤溶酶的深入研究,不仅有助于阐明蛇伤中毒患者的凝血病理机制,而且为其开发应用提供了理论基础。文章综述蛇毒纤溶酶的研究进展及应用前景,重点阐述其分子结构、酶学特性及其与出血活性的关系     

蛇毒纤溶酶Alfimeprase在大肠杆菌中的可溶表达和纯化

Alfimeprase是Fibrolase的突变体,是一种蛇毒纤溶酶,有纤溶活性而无出血性。根据Alfimeprase的氨基酸序列和大肠杆菌密码子偏爱性,利用PCR的方法合成Alfimeprase DNA序列,分别融合在NusA和MBP的C端,与分子伴侣FkpA在大肠杆菌Origami B(DE3)中共表达,融合蛋白NusA/Alfimeprase以部分可溶的形式存在,可溶部分占上清总蛋白的25%左右,通过镍柱亲合层析纯化和肠激酶切割得到具有纤溶活性的重组蛋白Alfimeprase。本研究是首次报道在大肠杆菌中可溶表达Alfimeprase,为以后深入研究其功能及应用奠定了基础。     

蛇毒纤溶酶的一般药理作用

目的研究蛇毒纤溶酶的药理作用.方法分别以低、中、高三个剂量组(5.0、10.0、20.0u/kg体重)研究蛇毒纤溶酶对麻醉犬颈动脉收缩压、舒张压、平均压、心率、心电图各参数、呼吸频率与幅度、凝血时间及复钙时间的影响.结果蛇毒纤溶酶(5.0、10.0、20.0u/kg)对麻醉犬血压、心率、心电、呼吸频率及呼吸幅度均无明显影响;低剂量(5.0u/kg)组蛇毒纤溶酶对犬凝血时间及复钙时间无明显影响,但中高剂量组(10.0、20.0u/kg)蛇毒纤溶酶可明显延长凝血时间及复钙时间,中剂量组对复钙时间的影响恢复较快;蛇毒纤溶酶对小鼠一般行为,机能协调功能及阈下戊巴比妥钠催眠作用无明显影响;对神经系统无明显影响.结论蛇毒纤溶酶能明显延长血液凝固时间及复钙时间,对循环系统、呼吸系统和神经系统无明显副作用.是一种安全有效的抗凝血药物.     

蝮蛇蛇毒纤溶酶对动物血栓形成的研究

目的探讨蝮蛇蛇毒纤溶酶对实验性动物血栓形成的影响.方法采用Chandler氏体外法形成大白鼠体外血栓和家兔半体内血栓,给药组和对照组分别注射蛇毒纤溶酶及相同体积的生理盐水,分别测定两组动物体外形成的血栓重量和长度.结果给药组动物形成的血栓明显小于对照组.结论蝮蛇蛇毒纤溶酶具有抑制血栓形成的作用.     

重组海蚯蚓纤溶酶的克隆、表达及活性鉴定北大核心CSCD

纤溶酶在溶栓治疗中起重要作用,能够溶解血凝块的主要成分纤维蛋白。采用RACE方法从海蚯蚓消化道组织中扩增出海蚯蚓纤溶酶编码序列,构建该基因原核表达载体,并进一步构建工程菌表达融合蛋白,经Ni2+树脂柱纯化后通过平板法检测该融合蛋白纤维蛋白酶原激活活性。结果获得海蚯蚓纤溶酶的c DNA序列和氨基酸序列,并成功构建重组表达载体p ET-21a-AFE,表达纯化出融合蛋白,该融合蛋白能够激活纤维蛋白酶原而溶解纤维蛋白。总之,本研究获得了海蚯蚓纤溶酶的c DNA序列和氨基酸序列,并初步证明其具有纤维蛋白酶原激活活性,为临床新型溶栓药物的开发提供实验基础。     

重组海蚯蚓纤溶酶的克隆、表达及活性鉴定

纤溶酶在溶栓治疗中起重要作用,能够溶解血凝块的主要成分纤维蛋白。采用RACE方法从海蚯蚓消化道组织中扩增出海蚯蚓纤溶酶编码序列,构建该基因原核表达载体,并进一步构建工程菌表达融合蛋白,经Ni2+树脂柱纯化后通过平板法检测该融合蛋白纤维蛋白酶原激活活性。结果获得海蚯蚓纤溶酶的c DNA序列和氨基酸序列,并成功构建重组表达载体p ET-21a-AFE,表达纯化出融合蛋白,该融合蛋白能够激活纤维蛋白酶原而溶解纤维蛋白。总之,本研究获得了海蚯蚓纤溶酶的c DNA序列和氨基酸序列,并初步证明其具有纤维蛋白酶原激活活性,为临床新型溶栓药物的开发提供实验基础。     

蛇毒纤维蛋白(原)溶解酶综述

蛇毒纤维蛋白溶解酶能直接溶解纤维蛋白(原).对蛇毒纤溶酶的深入研究,不仅有助于阐明蛇伤中毒患者的凝血病理机制,而且为其开发应用提供了理论基础.该文综述了蛇毒纤溶酶的研究进展及应用前景,重点阐述了蛇毒纤溶酶的分布和种类、分子结构及酶学特性等方面的内容.     

蛇毒纤溶酶的分离纯化-单克隆技术的成功应用

目的从蛇毒中纯化具有溶栓功效的纤溶酶.方法用DEAE-Sephadex A50离子交换,单抗亲和层析两步柱层析方法,对白眉蝮蛇蛇毒进行纯化,得到了一种纤溶酶活性组分.结果分离出的纤溶酶没有出血毒及神经毒等毒性,是一种安全的溶栓药物.结论运用单克隆技术的亲和层析能成功分离纤溶酶.     

蚯蚓纤溶酶基因P239的原核表达、纯化及活性测定

通过RT-PCR方法从蚯蚓(Lumbricus bimastus)中获得蚯蚓纤溶酶基因,命名为P-239,含有 852 个核苷酸,成熟肽编码 239 个氨基酸,通过Genbank 序列同源性比较,与已知的蚯蚓纤溶酶有一定的同源性,为首次获得的新基因.随后构建了pBV-220/P-239重组质粒,在大肠杆菌DH5α中进行原核表达,再经亲和层析柱纯化复性,其产物经活性测定,确定不仅具有激酶作用,而且具有直接溶栓活性.     

血栓病治疗药物-纤溶酶的生物来源

血栓病的干预方式包括抗血小板凝集、抗凝血和溶栓,对应临床应用的分别是抗血小板、抗凝血酶和纤溶酶溶栓药物;纤溶酶是主要的溶栓类药物,主要用于血栓发病后的治疗。纤溶酶种类多来自于生物体,更多来自于微生物的次级代谢。本文从作用方式、安全性和市场开发等方面总结了国内外纤溶酶的类别、研究现状和发展趋势,并对不同产纤溶酶的微生物菌株类别进行了总结,对不同来源的纤溶酶基因和氨基酸序列等信息进行了比较,发现不同物种的纤溶酶基因虽然有差异,但主要是丝氨酸蛋白酶类,氨基酸序列一致性比较高,说明丝氨酸蛋白酶活性功能区域是相对保守的。     

Investigation of the mechanism of temperature sensitivity of the electroreceptors of ampullae of lorenzini

In experiments on Black Sea skates (Raja clavata), the potential of the receptor epithelium of the ampullae of Lorenzini and spike activity of single nerve fibers connected to them were investigated during electrical and temperature stimulation. Usually the potential within the canal was between 0 and –2 mV, and the input resistance of the ampulla 250–400 k. Heating of the region of the receptor epithelium was accompanied by a negative wave of potential, an increase in input resistance, and inhibition of spike activity. With worsening of the animal's condition the transepithelial potential became positive (up to +10 mV) but the input resistance of the ampulla during stimulation with a positive current was nonlinear in some cases: a regenerative spike of positive polarity appeared in the channel. During heating, the spike response was sometimes reversed in sign. It is suggested that fluctuations of the transepithelial potential and spike responses to temperature stimulation reflect changes in the potential difference on the basal membrane of the receptor cells, which is described by a relationship of the Nernst's or Goldman's equation type.I. P. Pavlov Institute of Physiology, Academy of Sciences of the USSR, Leningrad. I. M. Sechenov, Institute of Evolutionary Physiology and Biochemistry, Academy of Sciences of the USSR, Leningrad. Pacific Institute of Oceanology, Far Eastern Scientific Center, Academy of Sciences of the USSR, Vladivostok. Translated from Neirofiziologiya, Vol. 12, No. 1, pp. 67–74, January–February, 1980.     

Determination of stoichiometry of radioiodination of proteins

A sensitive method for the detection of small quantities of hydrophobic antioxidant free radical scavengers such as butylatedhydroxytoluene (BHT) and butylatedhydroxyanisole (BHA) in aqueous samples is described. The procedure involves extraction of the hydrophobic free radical scavenger into an organic solvent phase, followed by the subsequent reaction of an aliquot of this extract with the stable cation radical tris(p-bromophenyl)amminium hexachloroantimonate (TBACA). In experiments with BHT and BHA, the loss of TBACA absorbance at 730 nm was found to be linearly proportional to the amount of antioxidant added, with quantities of BHT as small as 200 pmol being easily detectable. In aqueous suspensions of dimyristoylphosphatidylcholine vesicles, assays of the aqueous BHT concentration showed that BHT partitioned strongly into the membrane phase, achieving very high BHT/phospholipid ratios. For a given concentration of BHT, partitioning into the membrane phase was greater in large, multilamellar liposomes than in either small, single-walled vesicles or in purified rat brain synaptic vesicle membranes. Direct assay of BHT and BHA in phospholipid membranes, however, was complicated by a nonspecific interaction between TBACA and the phospholipid.