毕赤酵母中人rank胞外段的表达纯化及其阻断作用的分析-临床检验诊断学专业论文.docxVIP

PAGE PAGE 1 摘要 NF-κB 的受体活化因子配体/受体（RANKL/RANK）信号介导破骨细胞形成和 骨吸收。此外，此信号还可以影响其他生物学过程，包括免疫系统和癌症。 RANKL/RANK 信号在淋巴结发育、淋巴细胞分化、树突状细胞存活、T 细胞活 化和免疫耐受诱导中均扮演着重要角色。因此，抑制 RANKL 为阻止骨疾病、癌 症病人的骨转移、骨免疫相关疾病、打破中枢免疫耐受促进抗肿瘤免疫应答 T 细 胞的发生等提供一个新的思路与治疗手段。本研究首先对人 RANK 胞外段序列 进行毕赤酵母表达系统的密码子优化，然后克隆到 pPIC9K 酵母表达载体中，将 重组质粒电转化到毕赤酵母，筛选获得高表达 RANK 胞外段蛋白（RANK-N）的 菌株。我们通过 SDS、考马斯亮蓝染色和 Western Blot 鉴定，证实所构建 表达菌株及目的蛋白的正确性。经过摇瓶表达的超滤、Sephadex G-50 凝胶过滤 层析和 Q Sepharose FF 离子交换层析三个步骤纯化重组蛋白，获得了纯度在 95% 以上的目的蛋白。利用等离子共振（SPR）技术分析重组 RANK 蛋白与 RANKL 蛋白之间的亲和力，进而证实了重组 RANK-N 能结合 RANKL。通过一系列体外 体内实验发现重组 RANK-N 蛋白能够阻断 RANKL 激活的 RANK 信号。此外， 我们利用人结直肠癌小鼠移植瘤模型（patient derived xenograft，PDX）发现 RANK 胞外段蛋白可以抑制人结肠癌的生长。通过此项研究，我们建立了一个简单有效、 可线性放大的 RANK-N 表达纯化系统，为最终的大规模生产及成果转化奠定了 基础。 关键词：RANK，毕赤酵母，蛋白纯化，骨疾病，结肠癌 Abstract The role of the receptor activator of nuclear factor-κB ligand (RANKL)/RANK system is well characterized within bone, where RANKL/RANK signaling mediates osteoclastogenesis and bone resorption.However, this signaling has also been shown to influence biologic processes beyond the skeletal system, including in the immune system and in cancer. RANKL/RANK signaling is important in lymph-node development, lymphocyte differentiation, dendritic cell survival, T-cell activation, and the establishment of T cell central tolerance. Therefore, the blockade of the interaction between RANKL and RANK provides a new therapeutic approach to prevent bone diseases and immune mechanisms of bone-related diseases, to treat cancers that develop in the skeleton,to break the central immune tolerance for reducing tumor-specific effector T cell. In this study, the extra-cellular domain of human RANK (RANK-N) coding sequence, created with Pichia Pastoris favored codons, was cloned into pPIC9K vector, and the recombinant plasmid was transformed into Pichia Pastoris. The expression of RANK-N protein was confirmed by SDS, Coomassie brilliant blue stain and western blotting. Recombinant RANK-N protein was purified by a multiple step process includi