文档介绍：汕头大学
硕士学位论文
人CystatinC基因克隆、表达研究及其单克隆抗体和多克隆抗体
的制备
姓名:曾青红
申请学位级别:硕士
专业:免疫学
指导教师:李康生;苏绍波
2010-05-28
汕头大学医学院硕士学位论文
中文摘要
目的:构建重组质粒并原核表达人 Cystatin C,制备其鼠源性单克隆抗体和兔源性多克隆
抗体并鉴定其生物学活性,分析 Cystatin C 在食管癌和胃癌组织的表达分布情况。
方法:从人的胃癌细胞 AGS 上克隆扩增 Cystatin C 基因,构建 pET44-Cystatin C 重组质粒,
转化于大肠杆菌表达菌株 BL21(DE3)获得重组人 Cystatin C 蛋白。以复性后的纯化 Cystatin
C 包涵体蛋白免疫 BALB/c 小鼠和新西兰兔,从而制备鼠抗人 Cystatin C 单克隆抗体和兔抗
人 Cystatin C 多克隆抗体。经 Western Blot 和 ELISA 方法鉴定单克隆抗体及多克隆抗体的
特异性。应用免疫组化技术和 RT-PCR 等方法检测人 Cystatin C 在胃癌食管癌组织表达情况。
结果:成功构建了人 Cystatin C 重组质粒并获得其纯化原核表达蛋白。免疫印迹实验和
ELISA 实验提示所制备单克隆抗体和多克隆抗体对天然人 Cystatin C 有较高的特异性。
Cystatin C 蛋白在食管癌组织和胃癌组织表达升高相对于癌旁正常组织。其 mRNA 亦在肿
瘤组织中显示高表达
结论:成功构建 Cystatin C 原核表达载体,所纯化蛋白有良好的抗原性。成功制备高效价
的 Cystatin C 单克隆抗体和多克隆抗体。Cystatin C 在食管癌胃癌组织中表达升高。
关键词:Cystatin C;原核表达;蛋白纯化;单克隆抗体;多克隆抗体
I
汕头大学医学院硕士学位论文
Abstract
Objective: To construct a prokaryotic plasmid to express the human Cystatin C binant
protein and obtain anti-Cystatin C mAb/pAb by immunizing mice/rabbit, and to identify the
biological properties of the mAb/pAb. We prepared this project to detect the expression,
distribution of human Cystatin C in stomach neoplasms and esophageal cancer tissues.
Methods: The coding sequence of human Cystatin C was amplified by RT-PCR from human
gastric cancer AGS cell and cloned into the vector pET44, and then it was expressed in the E. coli
BL 21(DE3) to get Cystatin C binant protein. The refolding denatured purified Cystatin C
binant protein was injected to immunize the BALB/C mice to develop anti-Cystatin C mAb,
and immunized the rabbit to develop anti-Cystatin C pAb. Splenocytes of the immunized mice
were collected and fused with the mouse myeloma cell line NS-1 cells. ELISA, Western blot and
were used to detect specificity of mAb/pAb. The expression of Cystatin C in neoplasms and
esophageal cancer tissues was investigated by immunohistochemistry and RT-PCR.
Results: The prokaryotic plasmid