人RANKL胞外结构域原核表达载体的构建及表达条件优化

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。

The construction of prokaryotic expression vector and optimization of expression conditions of the extracelluar Domain of Human RANKL

The receptor activator of nuclear factor -kappa B ligand （RANKL） is an important member of the TNF superfami-ly, 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.