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题名	蛇毒血管内皮生长因子及丝氨酸蛋白酶类似物的研究
作者	吴健波
学位类别	博士
答辩日期	2007-06
授予单位	中国科学院研究生院
授予地点	北京
导师	熊郁良 ; 张云
关键词	菜花烙铁头蛇毒 圆斑蝰蛇 蝮蛇 竹叶青 山烙铁头 蛇毒血管内皮生长因子 丝氨酸蛋白酶 丝氨酸蛋白酶类似物
中文摘要	蝰科蛇毒中含有丰富的具有血管通透增强活性的蛋白组分，本论文结合生物化学与分子生物学手段对几种毒蛇的蛇毒血管内皮生长因子进行了研究。其中，从云南产菜花烙铁头（Trimeresurus jerdonii）蛇毒中分离得到一个血管内皮生长因子，TjsvVEGF，并研究了其活性与受体结合特性的关系。同时，对圆斑蝰蛇、蝮蛇、山烙铁头和竹叶青等几种蛇的svVEGFs进行了分子生物学研究。此外，我们还分离到了一个蛇毒丝氨酸蛋白酶类似物,TjsvSPH,还对该蛋白的理化性质进行了初步研究。 TjsvVEGF是一个表观分子量为29 kDa的二聚体蛋白，由两个相同大小的亚基组成。活性实验表明，它在10ng的剂量下即可明显提高血管通透性，活性强度与VEGF165相当。虽然TjsvVEGF的氨基酸序列与TfsvVEGF和Pm-VEGF具有较高的相似性，但是它们的受体结合特性却有很大差异。TjsvVEGF与VEGFR-1的结合能力很弱，而对VEGR-2有很高的亲和力。这说明，TjsvVEGF的活性主要是VEGFR-2介导的。最后，我们对导致TjsvVEGF对VEGFR-1低亲和力的原因进行了探讨。 利用PCR方法，我们从圆斑蝰蛇毒腺中得到了三种svVEGFs蛋白编码区长短不一的cDNA序列，其差异是由cDNA链中一段富含AG（3’拼接接受位点）的区段发生了核苷酸缺失产生的。蛋白编码区核苷酸的缺失导致其编码的三种svVEGF蛋白N末端的氨基酸序列和长短均产生较大差异。因此我们推测，蝰属svVEGFs蛋白N末端普遍较短可能是编码它们的mRNA前体对3’拼接点的不同选择产生的。同时，通过对cDNAs推导的氨基酸序列分析发现，蝮蛇svVEGF和山烙铁头svVEGF在与受体结合相关的多个重要位点上发生了氨基酸替换，提示它们是研究svVEGFs与VEGFR结合机制的良好材料。 通过分子筛、离子交换和亲和层析等方法，我们还从菜花烙铁头蛇毒中得到了一个不具有酶活性的丝氨酸蛋白酶类似物，命名为：TjsvSPH。内肽序列测定结果表明，TjsvSPH三联体结构中的组氨酸已突变为精氨酸，这很可能是导致其失去蛋白水解酶活性的主要原因。活性检测验表明，与许多已发现的蛇毒活性组分不同，TjsvSPH不具有蛋白水解酶活性，不能引起血小板聚集，也不抑制ADP和凝血酶诱导的血小板聚集。它对人血浆的复钙时间也没有影响。 TjsvSPH的氨基酸序列与典型蛇毒丝氨酸蛋白酶Halystase和Calobin有约77％的一致性。它与同科属来源的蛇毒丝氨酸蛋白酶类似物氨基酸序列相似性高达92％以上，与不同科属的也有约74％以上的相似性。通过对Genbank中六种毒蛇所有丝氨酸蛋白酶及其类似物进化分析，我们推测，蛇毒丝氨酸蛋白酶类似物很可能是由蛇毒丝氨酸蛋白酶进化而来，并在进化过程中形成了一类独特的蛋白质。
英文摘要	Viperid and crotalid contains a great variety of toxins which can increase vascular permeability. In present paper, several snake venom vascular endothelial growth factors (svVEGFs) were studied by biochemistry and molecular biology methods. A novel svVEGF, TjsvVEGF, was purified from the venom of Trimeresurus jerdonii, Its activity and receptor-binding properties were studied. At the same time, cDNA clones of svVEGFs were obtained from the venom gland of Vipera Russelli, Agkistrodon halys, Trimeresurus monticola, and Trimeresurus stejnegeri. What′s more, we purified and characterized a serine proteinase homolog, TjsvSPH, from Trimeresurus jerdonii venom. TjsvVEGF is a homodimer with an apparent molecular weight of 29 kDa. 10 ng TjsvVEGF remarkably increased vascular permeability in the skin of rats. Although the amino acid sequence of TjsvVEGF shares highly similar with TfsvVEGF and Pm-VEGF, their receptor-binding affinities are quite different. TjsvVEGF has a high affinity for VEGFR-2, but a very low affinity for VEGFR-1, suggesting that TjsvVEGF′s function is mainly mediated by VEGFR-2. And the reason why TjsvVEGF bound to VEGFR-1 weakly was discussed. Using PCR method, we cloned three types of cDNA coding for svVEGF from the venom gland of Vipera russelli russelli. Their lengths are various due to the loss of nucleotides in a region where closely clustered AGs, the 3′ splice site, are present. The different nucleotide deletions in mRNA lead to three kinds of svVEGF proteins with various amino terminal lengths and sequences. We assumed that the short amino terminal of svVEGFs from Viperinae subfamily should be the result of different selections of 3′ splice site of pre-mRNAs. We also found that both of the svVEGFs from Agkistrodon halys and Trimeresurus monticola have several substitutions in their possibly receptor-binding sites, which should raise to some interesting characteristics of their receptor selections. An inactive serine protease homolog, TjsvSPH, was purified from the venom of Trimeresurus jerdonii. The His in the catalytic triad of TjsvSPH has been changed to Arg and this should lead to its loss of proteolytic activity. Different from many known proteins from snake venom, TjsvSPH had no proteolytic activity. It could not induce human platelet aggregation or inhibit ADP/thrombin-induced platelet aggregation, or influence the recalcification time of human plasma. The primary structure of TjsvSPH shows about 77% identity to Halystase or Calobin. It has as high as 92% similarity with the svSPHs from the same family, and 74% identity to the ones from other families. From the phylogenetic analysis of all the snake venom serine proteases and their homologs from the six snake species in the Genbank, we presumed that snake venom serine protease homologs should evolve from snake venom serine proteases and have form a unique type of proteins.
语种	中文
公开日期	2010-10-14
内容类型	学位论文
源URL	[http://159.226.149.42:8088/handle/152453/6131]
专题	昆明动物研究所_动物活性蛋白多肽组学 昆明动物研究所_其他
推荐引用方式 GB/T 7714	吴健波. 蛇毒血管内皮生长因子及丝氨酸蛋白酶类似物的研究[D]. 北京. 中国科学院研究生院. 2007.

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