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茶树（Camellia sinensis）半胱氨酸蛋白酶抑制剂（Cystatin）抗体的制备与纯化摘要：Cystatins，半胱氨酸蛋白酶抑制剂，目前对其研究主要集中在Cystatin与肿瘤、抗虫、抑菌等方面。本课题利用大肠杆菌pET-32a原核表达系统表达茶树（Camellia sinensis）Cystatin蛋白，通过镍亲和层析柱纯化蛋白并透析，得到相对纯净的融合蛋白，大小约为31kDa。将融合蛋白作为抗原免疫小鼠制备多克隆抗体，免疫6周后静脉取血测抗血清效价。当效价达到1：64000时，对小鼠眼球取血获得抗血清，用Protein A Resin对抗血清进行纯化，再通过SDS-PAGE和ELISA检测纯化效果。SDS-PAGE显示抗体蛋白条带清晰，重链大小约50kDa，轻链大小约25kDa，杂带明显减少；ELISA测定显示纯化的抗体效价提升2倍以上，纯化效果良好。本实验为探究茶树Cystatin抗体的相关功能以及茶树与马陆（Prospirobolus joannsi）Cystatin序列相似性的原因奠定了基础。26566
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毕业论文关键词：Cystatin；抗体；纯化；效价；SDS-PAGE
Preparation and Purification of Tea Tree（Camellia sinensis） Cystatin Antibody
Abstract：Cystatins are a family of cysteine protease inhibitors. Current research of Cystatin is mostly related to tumor, insect-resistance and anti-microbial. In this study, tea tree（Camellia sinensis）Cystatin protein was expressed in E. coli pET-32a expression system. After, protein was purified throw High Affinity Ni-charged Resin and dialysis, and fusion protein about 31kDa was obtained. Subsequently, BALB/c mice were challenged with the fusion protein as antigen to get polyclonal antibody, and the titer of the antibody was measured after 6 weeks of immunization. After titer reached 1：64000, the antiserum was collected. The antiserum was purified by Protein A Resin and checked by SDS-PAGE and ELISA for purification effects. Finally, SDS-PAGE demonstrated that the immunoglobulin bands were clear, while irrelevant bands were not obvious. Heavy chain and light chain was about 50kDa and 25kDa, respectively. ELISA suggested that titer of purified antibody was improved more than 2 times compared with the unpurified. The purification was efficient, which makes contribution to investigating the function of Camellia sinensis Cystatin antibody and the similarity of Cystatin sequence between Camellia sinensis and Prospirobolus joannsi.
Key words: Cystatin; antibody; purification; titer; SDS-PAGE
目 录
摘要 1
关键词 1
Abstract 1
Key words 2
绪论 2
半胱氨酸蛋白酶抑制剂Cystatin简介 2
茶树Cystatin抗体制备简介 3
茶树Cystatin抗体纯化简介 4
1 材料与设备 6
1.1 试验材料 6
1.2 试验试剂 6
1.3 试验仪器 6
2 试验方法 6
2.1 原核表达茶树Cystatin蛋白 6
2.2 茶树Cystatin蛋白纯化与脱盐 7
2.2.1 His-Cystatin融合蛋白的亲和层析 7
2.2.2 融合蛋白His-Cystatin透析脱盐 7
2.3 融合蛋白His-Cystatin透析液冻干浓缩 8
2.4 免疫小鼠 8
2.4.1 抗原浓度测定——考马斯亮蓝法 8
2.4.2 抗原-福氏佐剂混悬液的制备 8
2.4.3 免疫及抗体的收集 8
2.5 Cystatin抗体纯化 9
2.5.1 Protein A Resin纯化抗体 9
2.5.2 纯化抗体冻干浓缩 9
2.5.3 纯化抗体检测 9
2.5.3.1 SDS-PAGE 9
2.5.3.2 ELISA间接法检测抗体效价 9