人sApo2L-Fc的分子构建及其在CHO细胞中的表达

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。

Construction and expression of human sApo2L-Fc fusion protein in CHO cells

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.