【作者】 蒋立平；

【导师】 梁宋平；

【作者基本信息】 湖南师范大学 ， 生物化学与分子生物学， 2008， 博士

【摘要】 虎纹捕鸟蛛(Ornithoctonus huwena)是分布在我国云南和广西省的一种毒蜘蛛。它的毒液是由许多生物活性成份组成的复杂混合物,通过蛋白质组学和多肽组学方法从粗毒中已经鉴定了90种蛋白质和47种多肽,但这仅仅为粗毒中的部分高丰度组份。为了克隆虎纹捕鸟蛛毒腺中编码多肽和蛋白质的基因,我们使用表达序列标签(EST)技术,构建了毒腺的定向全长cDNA文库。随机测序后获得468个EST,通过聚类分析,可分成69个族：24个族是重叠群(每个群包括2个EST以上)和65个族是单态(每个族只包括1个EST)。通过生物信息学分析,其中68%的EST属于的毒素转录子；13%的EST属于细胞转录子；19%的EST属于新的转录子。所有毒素转录子编码67个开放读码框(ORF),即67个前体肽。根据前体肽序列的差异,除了HWTX-XI和HWTX-XIII外,它们能被分成8个超家族：即HWTX-Ⅰ超家族、HWTX-II超家族、HWTX-X超家族、HWTX-XIV超家族、HWTX-XV超家族、HWTX-XVI超家族、HWTX-ⅩⅦ超家族和HWTX-ⅩⅧ超家族。67个前体肽能被推导出43个成熟肽,序列中含半胱氨酸残基丰富,因此命名为半胱氨酸结毒素(CKT),其中31个是新的成熟肽。非毒素转录子编码41个蛋白质,它们是细胞蛋白质或别的非CKT分子,用真核生物正向同源群(KOG)和基因本体论(GO)注释了这些蛋白质。通过毒腺转录组学和毒液蛋白组学比较发现：仅仅15个CKT分子同时存在于毒腺转录组和毒液蛋白质组中；29个编码CKT的转录子只在转录组中被发现,而它们的翻译产物在毒液蛋白质组中没有被发现。用两种方法却没有鉴定到一个相同分子量的细胞蛋白质或其它毒液成分。此外,首次在蜘蛛毒腺中发现了一个编码EF-hand蛋白质(命名为HWEFHP1)的转录子重叠群,它功能可能与毒素从毒腺组织分泌到毒液有关。当前关于蜘蛛毒素基因组DNA的报道很少。为了深入的研究虎纹捕鸟蛛中编码毒素的基因组DNA是否具有普遍的无内含子特性,我们根据cDNA序列设计特异或简并的引物,克隆并分析了编码三个超家族毒素(HWTX-XI超家族、HWTX-ⅩⅦ超家族和HWTX-ⅩⅧ超家族)的DNA序列,结果发现它们的基因组DNA中都不存在内含子。此外,也从基因组DNA中克隆到19个编码毒素的新基因。为了尽可能多的鉴定到HWTX-XI的亚型,我们设计了简并引物,进行了定向PCR、克隆和进化分析。克隆到编码38个HWTX-XI前体肽亚型的cDNA序列,这是虎纹捕鸟蛛中成员最多的超级族,推导形成31个成熟肽；HW11c21、HW11c25、HW11c40、HW11c50和HW11c10等在HWTX-XI的进化上可能具有重要意义。随着环境的改变,蜘蛛的种类和数量越来越少,仅靠天然资源远不能满足对毒素的全面研究和开发利用的需要。采用表达质粒pVT102Uα在酿酒酵母S78中表达HWTX-XI的产率达到每升12.5mg。为了探讨这个表达系统是否能成一个表达蜘蛛毒素的通用系统,我们克隆并表达了20个编码毒素样多肽的基因。最后,成功的表达出11个毒素基因。对这些表达产物进行了初步的动物水平和细胞水平的功能研究。HW11c4在10μM的浓度下能抑制约50%的Kv1.1电流；HWTX-ⅩⅦbl在10μM的浓度下对大鼠DRG细胞上的高电压激活的钙通道有明显的抑制作用；HW11c4、HWllc27和HW11c27m对trypsin有很强的抑制作用,且对chymotrysin和thrombin也有明显的抑制作用；其它的表达产物活性很弱或者还没有找到生物学活性。此外,为从虎纹捕鸟蛛毒腺中筛选到与电压门控钠离子通道结合的因子,我们分别构建了虎纹捕鸟蛛的毒腺酵母双杂交cDNA文库和Nav1.8胞外区的Bait质粒,并进行了初步筛选。综上所述,利用分子生物学、生物信息学、生物化学、质谱和膜片钳等技术,我们对虎纹捕鸟蛛毒腺的转录组和基因组DNA进行了研究,克隆了一批有价值的新基因,表达并初步分析了4个有重要应用前景的毒素分子的功能。更多还原

【Abstract】 The Chinese bird spider Ornithoctonus huwena distributed in the hilly areas of Yunnan and Guangxi province in southern China is a venomous spider. The venom of O. huwena is a mixture of various components with different types of biological activities. In the previous work of this lab, using proteomic and peptidomic methods,90 proteins and 47 peptides have been identified from the spider venom, but usually only the most abundant components have been analyzed. In order to clone the genes encoding the proteins and peptides in the venom glands, a directional cDNA library of O. huwena venom glands was constructed by using the expressed sequence tag (EST) strategy and 468 ESTs were generated by a random sequencing. All ESTs were grouped into 24 clusters and 65 singletons, of which 68.0% of total ESTs belong to toxin-like genes,13.0% are cellular transcripts and 19.0% have no significant similarity to any known genes. Precursors of all toxin-like genes can be classified into eight superfamilies (HWTX-Ⅰsuperfamily, HWTX-Ⅱsuperfamily, HWTX-Ⅹsuperfamily, HWTX-ⅩⅣsuperfamily, HWTX-ⅩⅤsuperfamily, HWTX-ⅩⅥsuperfamily, HWTX-ⅩⅦsuperfamily, HWTX-ⅩⅧsuperfamily) except HWTX-Ⅺand HWTX-ⅩⅢ, according to the identity of their precursor sequences. All precursors were processed to achieve 43 mature peptides with abundant cysteines, named cystine knot toxins (CKTs), and 31 of them were novel CKTs. All cellular proteins or other possible venom components were annotated by KOG (eukaryotic orthologous group) and GO (gene ontology) terms. Comparison of the CKT repertoire and proteins revealed through a proteomic versus a transcriptomic approach, only 15 CKTs were identified by both approaches,29 transcripts coding for CKTs were found in the transcriptome but not as translated proteins in the venom proteome. However, no cellular proteins with identical molecular weights was identified by both approaches. In addition, a cellular transcript contig coding for an EF-hand protein (named HWEFHP1) had been identified for the first time from spider venom glands, which might be involved in the secretion of toxins from the gland into the venom.The genomic DNA of toxins from the spider O. huwena is seldom reported. To further investigate whether the genes encoding toxin-like peptides contain a common intronless feature, the genomic DNA encoding toxins of three superfamilies were cloned by using sequence specific or partially degenerate primers based on their cDNA sequences. An unexpected finding was that the intron was lacking in the genomic sequences of three superfamilies. In addition, we have cloned and analyzed 19 novel genes encoding toxin-like precursors by using the genomic DNA of the spider O. huwena. In order to indentify more HWTX-XI isoforms,38 cDNAs encoding HWTX-XI superfamily precursor were cloned by using partially degenerate primers and these precursors were processed to achieve 31 mature peptides. A phylogenetic tree was generated by using the neighbor-joining method, and HW11c21, HW11c25, HW11c40, HW11c50 and HW11c10 may play a very important role in the evolution relationship of HWTX-XI.With the changement of the enviroment, species and quantites of spiders decrease sharply, precise analysis and application of peptide toxins from spiders are frequently restricted because of the difficulties of obtaining sufficient venom material. The expression of HWTX-XI was successful by using the plasmid pVT102U/a in the Saccharomyces cerevisiae strain S78, and the yield reached 12.5mg/L. In order to investigate whether this expression system is versatile to express the spider toxin genes, we clone and express 20 genes encoding toxin-like peptides. At last,11 toxin genes were successfully expressed. The functions of these expressed products were also identified. The IC50 value of HW11c4 is 10.0μM for the Kv1.1 channel. HWTX-ⅩⅦb1 can reduce the peak currents of high voltage activated Ca2+ channels on adult rat dorsal root ganglion (DRG) neurons. HW11c4, HW11c27 and HW11c27m are potent trypsin inhibitors and can also inhibit the chymotrysin and thrombin. However, the functions of other expressed products are very low or are not found at all.In addition, in order to get the molecules from the venom glands of the spider O. huwena interacting with voltage-gated sodium channels, a yeat two-hybrid cDNA library and 4 bait plasmids of Nav1.8 were constructed respectively, and then were screened.In summary, by using molecular biology, bioinformactics, biochemistry, mass spectra and patch-clamp, etc, we have investigated transcriptomes and genomic DNAs of the O. huwena venom glands. We also have cloned and expressed some novel genes, and then analyzed the functions of the expressed products.更多还原