| 题名 | 大蹼铃蟾白介素分子的全长克隆及Caspase-1多克隆抗体的制备 |
| 作者 | 王宣 |
| 学位类别 | 硕士 |
| 答辩日期 | 2013-05 |
| 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 | 大蹼铃蟾 IL-8 IL-18 Caspase-1 RACE 抗体制备 |
| 其他题名 | The Full-length Amplification of Two Interleukin Molecules of Bombina maxima and Polyclonal Antibody Preparation of Caspase-1. |
| 学位专业 | 生物工程 |
| 中文摘要 | 本论文分为两个部分。第一部分为大蹼铃蟾两个白介素分子的全长扩增，第二部分为大蹼铃蟾Caspase-1的多克隆抗体制备。 第一部分，我们利用cDNA末端快速扩增（RACE）技术对大蹼铃蟾IL-8和两条IL-18分子进行了末端扩增。白介素分子是脊椎动物体内普遍存在的一类细胞因子，参与机体抵抗外部侵略，调节免疫应答，介导炎症反应，在维持机体稳态，保持机体健康方面起着非常重要的作用。我们首先利用分子生物学手段从大蹼铃蟾组织中提取RNA，再用RACE相关试剂在RNA末端接上一个接头并反转录，利用已知片段的特异性引物和所接接头的互补性引物对合成的一链进行扩增，即可得到目标片段的末端序列。经过实验，我们得到了大蹼铃蟾IL-8和IL-18的编码区全长序列，并对所得序列进行了物种间的比对和分析。 此部分研究结果为今后的大蹼铃蟾免疫学研究提供了序列数据。 第二部分，我们制备了大蹼铃蟾的Caspase-1的多克隆抗体，即兔抗血清。Caspases又叫做cysteine-aspartic proteases或者cysteine-dependent aspartate-directed proteases（半胱氨酸-天冬氨酸蛋白酶），是一个半胱氨酸蛋白酶的家族，其在细胞凋亡、坏死和炎症反应中起着非常重要的作用。caspase-1是炎症相关的酶，可以在炎症小体组建过程中被活化，活化后的caspase-1具有激活IL-1β和IL-18的功能，从而启动免疫应答，参与免疫反应。我们首先利用原核表达系统获得了大蹼铃蟾Caspase-1的重组蛋白，再将此蛋白作为抗原刺激新西兰兔，获得了兔抗血清。利用生物化学手段对获得的抗血清进行了定性和定量分析，最终制备了高特异性高效价的兔抗大蹼铃蟾Caspase-1多克隆抗体。该抗体的制备为大蹼铃蟾炎症小体的研究提供了实验材料，为进一步研究炎症小体的形式和功能执行提供了基础。 |
| 英文摘要 | This thesis is divided into two parts. The first part is the full-length amplification of two interleukin molecules of Bombina maxima. The second part is the polyclonal antibody preparation of caspase-1 in Bombina maxima. The first part, we amplified the cDNA ends of IL-8 and two IL-18 molecules of Bombina maxima using rapid amplification of cDNA ends (RACE). Interleukins are a type of cytokines commonly found in vertebrate involved in the resistance to routine microbial flora, modulating the immune response and mediating inflammatory response. They play a very important role in maintaining a homeostatic balance and the health of the body. First, we extracted RNA from Bombina maxima organizations using molecular biology means, then connected an adaptor to the end of the RNA, at the same time, reverse transcript it. Finally, using the known fragment specific primer and the adaptor complementary primer to PCR, the full-length of a chain can be obtained. Through experiments, we got the full-length of IL-8 and IL-18 coding region of Bombina maxima. And compared the resulting sequences and with other species. The findings in this part supplied sequence data for future immunological studies of Bombina maxima. The second part, we prepared polyclonal antibody of Bombina maxima caspase-1(that is rabbit antiserum). Caspases is named as cysteine-aspartic proteases or cysteine-dependent aspartate-directed proteases. It is a cysteine proteases family which plays a very important role in apoptosis, necrosis and inflammation. Caspase-1 is an enzyme related to inflammation that can be activated in the process of the formation of a inflammasome. Activated Caspase-1 can activate an IL-1beta or IL-18, in which way to initiate the immune response and involve in the immune response. For using of the E.coli expression system we first got Caspase-1 recombinant protein which then be used as an antigen to stimulate the New Zealand rabbit for rabbit antiserum. We did qualitative and quantitative analysis for the obtained antiserum by means of biochemistry. Finally, we got high-specific and high titer polyclona antibody. The preparation of the antibody provided experimental materials for the study of Bombina maxima inflammasome and a basis for further study of the form and function of the inflammasome. |
| 语种 | 中文 |
| 公开日期 | 2013-06-08 |
| 内容类型 | 学位论文 |
| 源URL | [http://159.226.149.42:8088/handle/152453/7458]  |
| 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 | 王宣. 大蹼铃蟾白介素分子的全长克隆及Caspase-1多克隆抗体的制备[D]. 北京. 中国科学院研究生院. 2013. |
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