茶树（Camellia sinensis）半胱氨酸蛋白酶抑制剂（Cystatin）抗体的制备及纯化
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