眼镜王蛇泛素融合蛋白和核糖体蛋白L30的分子克隆与分析及大蹼铃蟾膜联蛋白A2样蛋白的纯化与活性研究
文献类型：学位论文
| 作者 | 韦双双 |
| 学位类别 | 硕士 |
| 答辩日期 | 2005-06 |
| 授予单位 | 中国科学院研究生院 |
| 授予地点 | 北京 |
| 导师 | 张云 |
| 关键词 | 眼镜王蛇 泛素融合蛋白 核糖体蛋白 大蹼铃蟾 膜联蛋白AZ 血小板 |
| 中文摘要 | 本论文分为两个部分。第一部分利用分子生物学手段对广西产眼镜王蛇（OPhiophagushannah）泛素融合蛋白基因和核糖体蛋白L30基因进行了克隆及分析；第二部分利用生物化学手段对大蹼铃蟾（Bombinamaxima）膜联蛋白A2样蛋白进行了初步研究。首先，从广西产眼镜王蛇毒腺中抽提总RNA，经mRNA纯化后构建眼镜王蛇毒腺。DNA文库，得到大概1.8*105个独立的克隆。从所构建的cDNA文库中，我们随机筛选200个克隆测序，得到两个在进化上高度保守的基因：泛素融合蛋白基因（GenBahk登录号为AF297036）和核糖体蛋白L30基因（GenBank登录号是AF297033）。前者cDNA的开放阅读框为387bp，后者为348bp。前者编码128个氨基酸残基组成的泛素融合蛋白前体；后者编码1巧个氨基酸残基组成的核糖体蛋白L30前体。由cDNA序列推导出的氨基酸序列分析表明，泛素融合蛋白前体包括N-末端的泛素结构域（76个氨基酸残基）和C-末端的核糖体蛋白L40结构域（52个氨基酸残基）。该蛋白为一高碱性蛋白，C末端含有一个"锌指"结构域。与16个物种比较的结果表明，眼镜王蛇与脊椎动物的泛素融合蛋白氨基酸序列相似度较高，具有高度的保守性。我们克隆到的眼镜王蛇泛素融合蛋白基因和核糖体蛋白L30基因为泛素家族和核糖体家族提供新的序列信息，有助于深入研究泛素蛋白和核糖体蛋白的结构、功能以及与其他物种的类似分子之间的相互关系。其次，通过阴离子交换，凝胶过滤和阳离子交换层析我们从大蹼铃蟾皮肤匀浆物中纯化了一个表观分子量为33000Da的单链蛋白，N-末端序列比较分析显示它与脊椎动物膜联蛋白AZ亚群有较高的同源性，与来自非洲爪蟾、红色原鸡和人膜联蛋白AZ序列的annexin核心的第一个重复区结构域序列一致性分别为78.9％、89％和68％，因此命名为大蹼铃蟾膜联蛋白AZ样蛋白（BAllLP）。BAllLP具有钙依赖性的抑制专一性血小板膜受体GPVI激动剂一Stejnulxin诱导人血小板聚集的生物学功能。 |
| 英文摘要 | 外文摘要 In this work, ubiquitin fusion protein and ribosomal protein L30 from a constructed Ophiophagus hannah venom gland cDNA library were cloned and compared with other ubiquitin fusion proteins and ribosomal protein L30s from different species. Meanwhile, purification and characterization of an annexin A2-like protein from Chinese red belly frog Bombina maxima skin were also reported. Firstly, total RNA was extracted from the venom gland of snake O. hannah. A king cobra venom gland cDNA library was then constructed using purified mRNA from the total RNA. Approximately 1.8 x 105 independent clones were got in this venom gland cDNA library. By random sequencing of 200 independent clones, two conserved cDNA sequences of house-keeping gene were obtained. One is ubiquitin fusion protein (GenBank accession number: AF297036), the other is ribosomal protein L30 (GenBank accession number: AF297033). The former has an open reading frame of 387 bp, encoding for a 128 amino acids ubiquitin fusion protein precursor composed of a 76 amino acids ubiquitin domain and followed by a 52 amino acids ribosomal protein L40 domain. The deduced precursor protein from the nucleotide sequence is basic. The C-terminus of the ubiquitin fusion protein contains the zinc-finger motif The latter has an open reading frame of 348 bp, encoding for 115 amino acids ribosomal protein L30 precursor. The predicted amino acid sequence of ubiquitin fusion protein is highly conserved when compared to the sequences of homologous proteins from diverse species. Secondly, a single chain protein with apparent molecular mass of 33,000 Dalton was purified from skin of B.maxima by a combination of ion exchange and gel filtration chromatography steps. N-terminal amino acid sequence determination indicated that it is an annexin A2-like protein, thus designated as BAIILR The N-terminal amino acid sequence of BAIILP shares 78.9%, 89% and 68% identity with those of the first repeat of annexin core domain of annexin A2 from Africa claw toad, red junglefowl and human, respectively. The annexin A2 like protein (BAIILP) from B.maxina inhibits stejnulxin, a specific platelet agonist via GPVI, induced platelet aggregation in a Ca2+ dependend manner. |
| 语种 | 中文 |
| 公开日期 | 2010-10-15 |
| 源URL | [http://159.226.149.42:8088/handle/152453/6164]  |
| 专题 | 昆明动物研究所_动物活性蛋白多肽组学 |
| 推荐引用方式 GB/T 7714 | 韦双双 . 眼镜王蛇泛素融合蛋白和核糖体蛋白L30的分子克隆与分析及大蹼铃蟾膜联蛋白A2样蛋白的纯化与活性研究[D]. 北京. 中国科学院研究生院. 2005. |
入库方式： OAI收割
来源：昆明动物研究所
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