Production of chimeric recombinant single domain antibody-green fluorescent fusion protein in Chinese hamster ovary cells
Production of chimeric recombinant single domain antibody-green fluorescent fusion protein in Chinese hamster ovary cells
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DOI：
10.1089/hyb.2006.037
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发表时间：
2007-02-01
期刊：
影响因子：
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通讯作者：
Mohammadi, M.
中科院分区：
文献类型：
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作者：
Bazl, M. Rajabi;Rasaee, M. J.;Mohammadi, M.
纳米体如VHH在治疗和成像领域的应用受到越来越多的关注。本研究以绿色荧光蛋白(GFP)为报告基因，构建了一株稳定的中国仓鼠卵巢(CHO)基因工程细胞系，并利用两套表达载体分别在细胞内和细胞外表达单域抗体。通过限制性内切酶图谱和序列分析来检测构建物的质量。逆转录-聚合酶链式反应(RT-PCR)进一步检测基因转染率和蛋白表达。将转基因细胞接种于200 mU g/mL潮霉素培养基中，获得稳定的细胞株，其荧光活性可持续180天以上。用荧光显微镜、荧光光谱分析和酶联免疫吸附试验(ELISA)检测融合蛋白的表达。这一策略允许快速生产涉及VHH的重组荧光抗体，可用于各种实验，如成像和检测，其中需要一种初级标记抗体。
There is an increasing interest in the application of nanobodies such as VHH in the field of therapy and imaging. In the present study a stable genetically engineered cell line of Chinese hamster ovary (CHO) origin transfected using two sets of expression vectors was constructed in order to permit the cytoplasmic and extracellular expression of single domain antibody along with green fluorescent protein (GFP) as reporter gene. The quality of the constructs were examined both by the restriction map as well as sequence analysis. The gene transfection and protein expression was further examined by reverse transcription-polymerase chain reaction (RT-PCR). The transfected cells were grown in 200 mu g/mL hygromycin containing media and the stable cell line obtained showed fluorescent activity for more than a period of 180 days. The production of fusion protein was also detected by fluorescent microscopy, fluorescent spectroscopy as well as by enzyme-linked immunosorbent assay (ELISA) analysis. This strategy allows a rapid production of recombinant fluobodies involving VHH, which can be used in various experiments such as imaging and detection in which a primary labeled antibody is required.