利用重组蛋白技术生产多克隆抗人金属硫蛋白2A抗体。

Production of the polyclonal anti-human metallothionein 2A antibody with recombinant protein technology.

作者信息

Marikar Faiz M M T, Sun Qi-Ming, Hua Zi-Chun

机构信息

The State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing 210093, China.

出版信息

Acta Biochim Biophys Sin (Shanghai). 2006 May;38(5):305-9. doi: 10.1111/j.1745-7270.2006.00167.x.

DOI:10.1111/j.1745-7270.2006.00167.x

PMID:16680370

Abstract

Metallothionein 2A (MT2A) is a small stress response protein that can be induced by exposure to toxic metals. It is highly expressed in breast cancer cells. In this study, the cDNA encoding the human MT2A protein was expressed as glutathione S-transferase (GST) fusion protein in Escherichia coli. Recombinant MT2A proteins were loaded onto 12% sodium dodecylsulfate-polyacrylamide gel and separated by electrophoresis, the recombinant protein was visualized by Coomassie blue staining and the 33 kDa recombinant GST-MT2A fusion protein band was cut out from the gel. The gel slice was minced and used to generate polyclonal antisera. Immunization of rabbit against MT2A protein allowed the production of high titer polyclonal antiserum. This new polyclonal antibody recognized recombinant MT2A protein in western blot analysis. This low-cost antibody will be useful for detection in various immuno-assays.

摘要

金属硫蛋白2A（MT2A）是一种小的应激反应蛋白，可由接触有毒金属诱导产生。它在乳腺癌细胞中高度表达。在本研究中，编码人MT2A蛋白的cDNA在大肠杆菌中表达为谷胱甘肽S-转移酶（GST）融合蛋白。将重组MT2A蛋白加载到12%的十二烷基硫酸钠-聚丙烯酰胺凝胶上并通过电泳分离，重组蛋白通过考马斯亮蓝染色可视化，从凝胶上切下33 kDa的重组GST-MT2A融合蛋白条带。将凝胶切片切碎用于制备多克隆抗血清。用MT2A蛋白免疫兔子可产生高滴度的多克隆抗血清。这种新的多克隆抗体在蛋白质免疫印迹分析中可识别重组MT2A蛋白。这种低成本抗体将有助于各种免疫检测。