目的:构建人sApo2L-Fc分子,在中国仓鼠卵巢细胞(CHO)中表达有生物学活性的人Apo2L-Fc融合蛋白。 方法:将sApo2L-Fc基因克隆入pcDNA3.1(+)表达载体,重组质粒转化大肠杆菌DH5α。挑取阳性克隆扩大培养,提取质粒进行酶切鉴定;采用脂质体法将重组质粒转入CHO细胞,经G418加压筛选、ELISA检测,挑选表达较高的阳性转化子扩大培养;表达的sApo2L-Fc融合蛋白经Protein A亲和柱纯化,纯化产物用SDS-PAGE、Western Blotting检测样品的分子量及免疫原性,用L929细胞进行生物活性测定。 结果:酶切鉴定及测序显示重组子构建与预想一致;ELISA证实了sApo2L-Fc融合蛋白在CHO细胞中的表达;SDS-PAGE检测到纯化产物的分子量与理论分子量相符;在同样的位置,Western Blotting显示阳性;L929细胞测定:纯化产物的生物学活性达1.0×105IU/mg。 结论:构建了sApo2L-Fc的表达载体,并成功地在CHO中表达,表达的sApo2L-Fc融合蛋白具有生物学活性。
Abstract: Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL, Apo-2L) is a mediator of cell death that preferentially targets cancer cells. The C-terminal extracellular region of Apo-2L (amino acids 114-281) exhibits a homotrimeric subunit structure. Soluble Apo-2L(sApo-2L) induces extensive apoptosis in lymphoid as well as non-lymphoid tumor cell lines. But it is limited in its efficacy by the short circulating half-life and low stability. sApo2L-Fc is a fusion protein consisting of Apo-2L and a fragment(hinge, CH2, CH3 domains) of the Fc of the IgG1. The DNA was constructed in the pcDNA3.1(+) eukaryotic expression vector. Using DOTAP-mediated gene transfer technique, pcDNA3.1- sApo2L-Fc was transformed into Chinese hamster ovary(CHO) cells. The selective medium containing G418 was then employed to select the positive colony. After purified with protein A affinity chromatography, the product was detected with SDS-PAGE and Western blot. A protein band with molecular weight of 44KDa was found in the SDS-PAGE. Western blot displayed the recombinant product had strong immunological activity with mouse anti-human Fc monoclonal antibody. After purification, the biological activity of sApo2L-Fc was more than 1.0×105IU/mg.
牛晓霞,田雅峰,刘金毅,吴晓东. 人sApo2L-Fc的分子构建及其在CHO细胞中的表达[J]. 中国生物工程杂志, 2008, 28(2): 21-24.
. Construction and expression of human sApo2L-Fc fusion protein in CHO cells. China Biotechnology, 2008, 28(2): 21-24.
https://manu60.magtech.com.cn/biotech/CN/ 或 https://manu60.magtech.com.cn/biotech/CN/Y2008/V28/I2/21
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