【摘要】 细胞核因子κB受体活化因子配体(Receptor activator of the NF-κB ligand,RANKL)是TNF超家族的重要成员之一,属于Ⅱ型跨膜蛋白,通过与其受体核因子κB受体活化因子(RANK)构建的信号通路参与乳腺癌的发生、肿瘤骨转移、骨质疏松、关节炎等病理过程。人RANKL胞外结构域基因片段与原核表达载体pET-21b融合并转化至表达菌Rosetta,并对其表达条件进行了优化。通过对诱导时机,诱导温度,异丙基-β-D-硫代吡喃半乳糖苷(IPTG)浓度,诱导时间及甘油浓度的条件优化表明,当IPTG的浓度为0.2 mmol/mL,20℃振荡诱导培养6 h时,甘油浓度为4%时,可在上清中获得高效表达的pET-21bRANKL融合蛋白,为该蛋白的进一步纯化及结构与功能研究打下了良好的基础。更多还原

【Abstract】 The receptor activator of nuclear factor-kappa B ligand( RANKL) is an important member of the TNF superfamily,which is a type II transmembrane protein,and its cognate ligand,RANK( receptor activator of nuclear factor-kappa B)built the signaling pathway in the occurrence of breast cancer,bone metastases,osteoporosis,arthritis and other pathological processes. In this article,the extracellular domain of human RANKL gene were cloned into prokaryotic expression vector pET-21b and transformed into the expression bacteria E. coli Rosetta. We successfully constructed recombinant human RANKL gene Rosetta / pET-21b-RANKL. And it was induced to express the target protein. We optimized its expression conditions.When the IPTG concentration was 0. 2 mmol / mL,the induced timing OD600 for 0. 6,oscillation induced culture for 6 h in 20℃ and glycerol concentration for 4%,we can efficiently expressed the extracellular domain of human RANKL fusion protein( pET-21b-RANKL) in the supernatant. It provided a foundation for further purification of the protein and researched its structure function.更多还原