【作者】 袁春华；

【导师】 梁宋平； 谢锦云；

【作者基本信息】 湖南师范大学 ， 生物化学与分子生物学， 2003， 硕士

【摘要】 1：虎纹捕鸟蛛（Ornithochoctonus huwena）是分布在我国西南部山区的一种蜘蛛新种。它的粗毒中含有多种活性成分，其中研究的比较深入的多为神经毒素，如HWTX-Ⅰ，这是一种突触前膜N型钙离子通道阻断剂；毒素HWTX-Ⅳ，它是一种TTX敏感型钠离子通道阻断剂；另外，还从虎纹粗毒中纯化出一种具有凝集活性毒素成分SHL-Ⅰ。 本文报道从虎纹捕鸟蛛（Ornithochoctonus huwena）粗毒中，应用阳离子交换和反相高效液相色谱的方法分离到一种胰蛋白酶抑制剂，命名为Huwentoxin-Ⅺ（HWTX-Ⅺ）。经MALDI-TOF质谱技术鉴定，HWTX-Ⅺ的相对分子质量为6166.2。经DTT还原，进一步用碘乙酰胺修饰其相对分子质量增加到6514.2，根据一个半胱氨酸修饰相对分子质量增加58，我们可以推断HWTX-Ⅺ有三对二硫键。利用Edman降解气相蛋白质测序仪测得HWTX-Ⅺ的一级结构NH2-IDTCRLPSDRGRCKASFERWYFNGRTCAKFIYGGCGGNGNKFPTQEACMKRCAKA-COOH，理论相对分子质量比质谱测得的值多6，由此也可以确定六个半胱氨酸形成了三对二硫键。利用传统的酶促动力学研究方法通过分光光度计测定HWTX-Ⅺ对胰蛋白酶的抑制活性发现HWTX-Ⅺ对胰蛋白酶有很强的抑制作用，完全抑制的摩尔抑制比为1：1。用Lineveaer-Burk作图法得知，该抑制剂属竞争性抑制类型，具ki值为2.938×10^-8mol/L（以BAPNA为底物）。用BIAcore X测定该毒素和胰蛋白酶及胰凝乳蛋白酶的生物大分子相互作用，同样发现HWTX-Ⅺ对胰蛋白酶有很强的抑制作用，对胰凝乳蛋白酶也有中等程度的抑制作用，解离常数Kd分别为1.23×10^-10mol／L和1.14×10^-7mol/L。HWTX-Ⅺ对昆明种小白鼠第四脑室注射表现出较强的活性，其半致死量LD₅₀为256ug／kg。由此可见，HWTX-Ⅺ也是一种哺乳动物中枢神经毒素。更多还原

【Abstract】 Abstract l:The Chinese bird spider, Ornithochoctonus huwena, distributed in the hilly areas of Yunnan and Guangxi in the south of China, was recently identified as a new species and is one of the most venomous spiders in China. In our previous work, we have demonstrated that 0. huwena venom contains a mixture of compounds with different types of biological activities. Most of them are neurotoxins, such as HWTX-1, -II ,-III, IV.Huwentoxin XI (HWTX~XI),a serine protease! inhibitor, consists of 55 amino acid residues with three disulfide bridges. The toxin was isolated from the venom of the Chinese spider Ornithochoctonus huwena by ion-exchange chromatogram -phy and reverse phase high performance liquid chromatography.The jmolecular weight of HWTX-XI is 6166. 2, determined by MALDI-TOF mass spectrometry ,which is identical with thecalculated mass based on the complete amino acid sequence of NH2-IDTCRLPSDRGRCKASFERWYFNGRTCAKFIYGGCGGNGNKFPTQEACMCAKA - COOH, determined by automatic Edman degradeation .The inhibition of trypsin by HWTX-XI was competetive ,with a ki value of 2. 938 X 108mol/L (BAPNA as substrate), which is determined by using a U-2000 spectrophotometer instrument (HITACHI , Japan) .The binding properties of HWTX-XI on trypsin and a -chymotrypsin were measured by a BIAcore binding assay system. Results showed that HWTX-XI could bind to trypsin with a dissociation constant of 1. 23 X 10-10mol/L, and to a -chymotrypsin with a dissociation constant of 1. 14 X 10-7mol/L . Inhibition profiles of trypsin by HWTX-XI revealed a 1:1 binding stoichiometry between this inhibitor and trypsin.Huwentoxin XI also have a strong biological activity with a LD5n=256 u g/kg when injected into the fourth ventricle of the adult mouse brain.更多还原