中国组织工程研究

中国组织工程研究 ›› 2012, Vol. 16 ›› Issue (42): 7877-7882.doi: 10.3969/j.issn.2095-4344.2012.42.017

• 组织构建实验造模 experimental modeling in tissue construction • 上一篇    下一篇

抗人凋亡诱导因子单克隆抗体的制备与鉴定

戴启宇1，邓 昆1，王会英1，刘 宇2，杨廷桐1，陆建福1，钱 磊1   

  1. 1解放军笫371中心医院检验病理科，河南省新乡市 453000 ；2新乡市中心医院妇产外科，河南省新乡市 453000
  • 收稿日期:2012-07-09 修回日期:2012-08-16 出版日期:2012-10-14 发布日期:2012-10-14
  • 通讯作者: 邓昆，在读博士，主管技师，解放军笫371中心医院检验病理科，河南省新乡市 453000 drdengkun@gmail.com
  • 作者简介:戴启宇，男，1954年生，主任技师，主要从事临床生物化学、分子生物学方面的研究。 DQY 716@sohu.com

Preparation and identification of a monoclonal antibody against human apoptosis-inducing factor

Dai Qi-yu1, Deng Kun1, Wang Hui-ying1, Liu Yu2, Yang Ting-tong1, Lu Jian-fu1, Qian Lei1   

  1. 1Department of Inspection and Pathology, No. 371 Central Hospital of PLA, Xinxiang 453000, Henan Province, China; 2Department of Maternity Surgery, Xinxiang Central Hospital, Xinxiang 453000, Henan Province, China
  • Received:2012-07-09 Revised:2012-08-16 Online:2012-10-14 Published:2012-10-14
  • Contact: Deng Kun, Studying for doctorate, Technician in charge, Department of Maternity Surgery, Xinxiang Central Hospital, Xinxiang 453000, Henan Province, China Drdengkun@gmail.com
  • About author:Dai Qi-yu, Chief physician, Department of Inspection and Pathology, No. 371 Central Hospital of PLA, Xinxiang 453000, Henan Province, China DQY 716@sohu.com

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摘要：

背景：凋亡诱导因子不仅具有氧化还原和电子传递功能，还具有促细胞凋亡功能，从而在维持细胞正常生理活动中具有重要作用。
目的：制备抗人凋亡诱导因子单克隆抗体并进行生物学特性鉴定。
方法：利用Ensembl数据库及DNAstar软件包对凋亡诱导因子氨基酸序列进行分析，获得优势表位多肽，采用碳化二亚胺法将多肽与载体蛋白偶联制备完全抗原免疫动物，采用杂交瘤技术制备凋亡诱导因子单克隆抗体并纯化。
结果与结论：间接ELISA法检测显示，免疫鼠腹水中抗人凋亡诱导因子单克隆抗体效价达到1∶252 400。Western blot结果显示，存在与抗原带一致的相对分子质量67 000的特异条带。免疫组化检测结果显示，在结肠癌组织细胞中有棕色阳性颗粒表达，说明此抗体也可用于免疫组识化学染色。提示实验获得了高活性、高纯度、高特异性抗人凋亡诱导因子单克隆抗体。

关键词: 凋亡诱导因子, 杂交瘤细胞, 单克隆抗体, 凋亡, 抗体制备, 活性, 纯度, 特异性, 结肠癌, 组织工程

Abstract:

BACKGROUND: Apoptosis-inducing factor not only has the functional redox and electron transfer function, but also has a pro-apoptotic function, so it plays an important role in maintaining the normal physiological activity of the cells.
OBJECTIVE: To prepare mouse anti-human apoptosis-inducing factor monoclonal antibodies and to identify their biological characteristics.
METHODS: The Ensembl database and DNAstar package were used to analyze amino acid sequence of apoptosis-inducing factor and obtained the epitope polypeptide. Carbodiimide method was used to prepare the complete antigen immunized animal through coupling the polypeptide with a carrier protein. The BALB/c mice were immunized with a designed apoptosis-inducing factor peptide (KLH-coupled), and then the anti-AIF McAb was obtained by means of the B lymphoma hybridoma technique. Hybridoma technique was used to prepare and purify the apoptosis-inducing factor monoclonal antibodies.
RESULTS AND CONCLUSION: The detection of indirect ELISA method showed that titer of apoptosis-inducing factor monoclonal antibodies in ascetic fluid reached 1: 252 400. The result of Western blot demonstrated that there was specific Mr 670 000 brand which consistent with the antigen band. Immunohistochemical detection results showed there was brown positive expression in the colon cancer cells, which indicated that the antibodies can also be used for immunohistochemistry staining. Hyper-activity, specific and purified mouse anti-human apoptosis-inducing factors have been successfully obtained and identified.

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