三种有毒动物蛋白的分离纯化、分子克隆和生物活性的研究
文献类型：学位论文
| 作者 | 曾琳 |
| 学位类别 | 博士 |
| 答辩日期 | 2011-06 |
| 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 眼镜蛇毒因子 黑尾胡蜂 磷脂酶 大蹼铃蟾 乳清酸 |
| 其他题名 | Isolation and cloning of three proteins from poison animals and studies on their bioactivities |
| 学位专业 | 动物学 |
| 中文摘要 | 为了适应环境,很多动物(如两栖类、爬行类和昆虫)在长期的进化过程中演化出了活性高、专一性强的蛋白和多肽。它们作用的特异性和专一性，使其成为蛋白质、多肽结构与功能研究的良好模型，并为研究人类自身生理、病理机制提供工具和线索。同时它们还是开发临床诊断试剂和治疗药物的天然宝库。本文以两栖类、爬行类、昆虫毒腺分泌物为研究对象，分离纯化到3个活性蛋白，并进行了分子克隆和生物活性的研究。 眼镜蛇毒因子（cobra venom factor, CVF）是分布于眼镜蛇科毒蛇毒液中的蛋白质。CVF是补体系统C3b的类似物，在血清中能够形成CVF,Bb(C3/C5转化酶)，持续高效的激活补体系统，并且不受补体系统的调控因子H因子和I因子的调节。不同来源的CVF形成的转化酶，切割C5的活性差距很大。CVF可以抑制器官移植后产生的免疫排斥反应，同时还可以用于免疫疾病致病机理的研究，并有希望开发成为相关疾病的治疗药物。人源化CVF构建已经引起关注。然而目前为止，仅有少数几个蛇种的CVF蛋白得到了纯化和活性研究，CVF cDNA仅见于Naja kaouthia和Austrelaps superbus。CVF蛋白的分离纯化、活性鉴定和分子克隆，可以为CVF相关药物的开发提供新结构和功能的信息。利用常压分离纯化柱Superdex G-100(superfine)，中压液相分离纯化柱Resource Q(1 ml)和Hitrap Heparin HP(1 ml)，我们从眼镜王蛇（Ophiophagus hannah）的毒腺分泌物中纯化到了眼镜王蛇的CVF，命名为OVF。OVF由α、β、γ 3个亚基组成，在SDS-PAGE中，非还原条件下为一条带，表观分子量约为140 KD；还原条件下为三条带，分子量大小分别为72 KD(α 亚基)、45 KD（β亚基）和32 KD（γ 亚基）。OVF为高分子量的糖蛋白，Schiff试剂染色表明，眼镜王蛇OVF的α链被糖基化。OVF能够抑制豚鼠血清对致敏的绵羊红细胞的裂解，暗示OVF能够消耗豚鼠血清中的补体成分；同时，OVF能够激活豚鼠血清对豚鼠红细胞的裂解，暗示OVF能够激活补体系统，形成攻膜复合体裂解细胞。对OVF各个亚基分别测序，根据已知的Naja kaouthia CVF和C3的cDNA序列，并结合N端测序的结果设计引物克隆得到OVF的cDNA。序列分析显示OVF与Naja kaouthia CVF，Austrelaps superbus AVF-1和AVF-2的氨基酸序列高度相似，表明在眼镜蛇科毒蛇中CVF是高度保守的。OVF生物活性的检测和cDNA的克隆为揭示CVF结构和功能的关系提供了新的信息。 磷脂酶是胡蜂毒液中主要的过敏原之一。此外，胡蜂毒液磷脂酶还具有溶血、致炎、诱导血小板聚集、诱导血栓、致死活性。我们从黑尾胡蜂（Vespa tropica）的毒液中分离到一个分子量32 KD的蛋白质，命名为Vtp32，N端序列比对和肽指纹图谱的结果暗示了Vtp32为磷脂酶。磷脂酶A1底物切割活性分析显示Vtp32 为具有PLA1底物识别活性。Vtp32处理的蛋黄卵磷脂具有溶血活性。Vtp32还具有抗凝活性，这在胡蜂磷脂酶中是第一次报道。 乳清酸蛋白(whey acidic protein, WAP)是一类含有“四二硫化物核心”(four disulphide core, FDC)结构域的蛋白质。乳清酸蛋白的FDC结构域也可称为WFDC (whey four disulphide core, WFDC) 结构域。通过大蹼铃蟾皮肤cDNA文库的构建和大规模测序，发现了10个编码乳清酸蛋白的克隆，含完整的开放式阅读框的克隆有6个，分为3类。多个编码乳清酸蛋白的克隆的发现暗示了在大蹼铃蟾皮肤中乳清酸蛋白是高度表达的。为了纯化大蹼铃蟾皮肤中乳清酸蛋白，并研究其在大蹼铃蟾皮肤中的生物活性和生理作用，我们利用pMal p2×原核表达系统表达了包含麦芽糖结合蛋白融合头的大蹼铃蟾WFDC结构域，并制备了抗WFDC抗血清、纯化了抗WFDC抗体。用纯化后的抗体在大蹼铃蟾皮肤分泌物和匀浆物中均检测到了特异性杂交条带。大蹼铃蟾抗WFDC结构域抗体的制备有利于大蹼铃蟾乳清酸蛋白的纯化和功能研究。 |
| 英文摘要 | During the long-term evolution，animals such as amphibians, reptiles and insects produce rich proteins and peptides in their venom gland secretions. These proteins show high activity and strong specificity towards their substrate, which provide perfect models for the research of protein structure and function. They can also be used as powerful tools to study the human pathogenesis of diseases and physiological mechanism. And they are also natural pools for the development of clinical diagnosis reagent and therapy drug. Cobra venom factor (CVF) is an anti-complement factor existed in cobra venom. It is a functional analogue of C3b. Like C3b, it binds factor B in the presence of Mg2+ ions. This new formed complex can be cleaved by factor D, then generates a complex CVF,Bb and a peptide Ba. CVF,Bb is a C3/C5 convertase which is resistant to the regulation of factor H and I. Different CVFs have different levels of C5 hydrolytic activity. CVF has been used as experimental tool to decomplement laboratory animals to study the functions of complement in host defense and immune response as well as in the pathogenesis of diseases. In the present work, a cobra venom factor termed OVF was purified and characterized from the venom gland secretions of Ophiophagus hannah by successive gel fltration, ion-exchange and heparin affinity chromatography. The purified OVF was homogenous on SDS-PAGE gel with an apparent molecular weight of 140 KDa under non-reducing conditions. Under reducing condition, OVF divided into three bands with apparent molecular weight of 72 KDa, 45 KDa and 32 KDa， respectively. OVF Consumed complement component with CH50 of 6.5 ug, and activated the bystander lysis of guinea pig erythrocytes. By RT-PCR and 5'RACE method, the full-length cDNA were also obtained. Result of MALDI-TOF assay indicated the cloned cDNA matched the OVF protein very well. Alignment of OVF’s deduced protein sequence with other known CVFs raveled that it is highly conserved. The alignment of CVF of all known species and cobra C3 reveal CVFs are high conservative molecules with identity above 80% between each other. The Phylogentic studies reveled OVF is closer to CVF from Naja kaouthia CVF than AVF-1, and AVF-2. In wasp venom, Phospholipase is a main allergen to human. However, the Phospholipases from wasp venoms also have other bioactivities including inducing hemolysis, inducing inflammation, activating platelet aggregation, inducing thrombosis in vivo and lethal activity. A protein named Vtp32 with molecular weight of 32 kDa was purified from the venom gland secretions of Vespa tropica by successive gel filtration and heparin affinity chromatography. Vtp32 showed PLA1 activity. Egg yolk lecithin treated by Vtp32 can lysis the erythrocytes. Beside Vtp32 has anti-coagulation activity, this is the first report about the anti-coagulation phospholipase in wasp venom. Whey acidic protein (WAP) is the protein with (four disulphide core, FDC) domain. The FDC domain of WAP can also called WFDC domain. In the cDNA library of Bombina maxima, we found ten clones that coding WFDC domain, six of which contain the entire open reading frame. So it implies that WAP is abundant in the skin secretion of Bombina maxima. In order to isolate Bombina maxima WAP and reveal it’s bioactivities, we expressed the WFDC domain of WAP with pMal p2×expression system. The fusion protein fuse with maltose-binding protein (MBP) is purified by amylase resin Column. The New Zealand big ear rabbit is inoculated by the purified protein and the polyclone antibody was collected and purified. The purified antibody can detect the expressed fusion prtotein. Proteins with molecular weight of 17 kDa in the Bombina maxima skin secretion were also detected by the purified antibody. So it is confirmed that WAP indeed exist in Bombina maxima skin secretion. |
| 语种 | 中文 |
| 公开日期 | 2013-04-24 |
| 源URL | [http://159.226.149.42:8088/handle/152453/7387]  |
| 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 | 曾琳. 三种有毒动物蛋白的分离纯化、分子克隆和生物活性的研究[D]. 北京. 中国科学院研究生院. 2011. |
入库方式： OAI收割
来源：昆明动物研究所
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