小鼠核因子κB受体活化因子配基（mRANKL）活性区的克

发布时间：2018-07-08 11:13

本文选题：小鼠核因子κB受体活化因子配基(mRANKL) + 基因克隆　；参考：《华东师范大学》2010年硕士论文

【摘要】： 核因子κB受体活化因子配基(RANKL)是诱导破骨细胞分化,成熟的重要因子,在生物学及医学研究中具有广泛的应用。RANKL主要结合到破骨前体细胞的核因子κB活化因子受体(RANK)上,启动下游NF-κB,P38,ERK,JNK等信号通路,刺激破骨细胞的分化,成熟及成熟后的破骨细胞存活。由于RANKL在破骨细胞的发育过程中发挥重要作用,所以它的失调可引发多种骨骼疾病,如骨质疏松,风湿性关节炎等疾病。现在开发了多种针对RANKL及它所调控的信号通路的药物,用于治疗RANKL所引发的骨骼疾病。因此建立制取高纯度的鼠源RANKL(mRANKL)重组蛋白,并诱导破骨细胞前体向破骨细胞分化这个模型具有重要的现实意义。它不仅可以为治疗骨骼疾病的筛药实验提供模型,而且可以极大的降低实验室成本。在本实验中,以鼠的骨髓细胞cDNA为模板,利用PCR技术体外扩增小鼠核因子κB受体活化因子配基(mRANKL)活性区cDNA,将PCR产物克隆至His标签的融合蛋白表达载体pET28a(+),鉴定正确的质粒转化至BL21表达菌中。通过不同温度、IPTG浓度及诱导时间诱导目的蛋白表达,筛选出mRANKL融合蛋白表达的最佳条件。以最佳条件大量纯化mRANKL重组蛋白,纯化后的蛋白过滤并稀释成不同浓度,与商业购买的mRANKL蛋白同时分别刺激小鼠单核／巨噬细胞Raw264.7细胞分化,可以看见有明显的破骨细胞形成,表明mRANKL重组蛋白具有生物活性,可替代商业产的鼠源RANKL。
[Abstract]:Nuclear factor 魏 B receptor activating factor ligand (RANKL) is an important factor in inducing osteoclast differentiation and maturation. Activation of downstream NF- 魏 B signaling pathway, such as P38 ERKN JNK, stimulated osteoclast differentiation and survival of mature and mature osteoclasts. Because RANKL plays an important role in the development of osteoclasts, its imbalance can lead to many skeletal diseases, such as osteoporosis, rheumatoid arthritis and so on. A variety of drugs have been developed for RANKL and its regulated signaling pathways to treat bone diseases caused by RANKL. Therefore, it is of great practical significance to establish a high purity rat RANKL (mRANKL) recombinant protein and induce osteoclast precursors to differentiate into osteoclasts. It can not only provide a model for screening drugs for treatment of bone diseases, but also greatly reduce the cost of laboratory. In this experiment, mouse bone marrow cell cDNA was used as template. The active region of mouse nuclear factor- 魏 B receptor activating factor ligand (mRANKL) was amplified by PCR technique in vitro. The PCR product was cloned into the fusion protein expression vector pET28a (), of his label and the correct plasmid was transformed into BL21 expression strain. The optimal conditions for the expression of mRANKL fusion protein were obtained by inducing the expression of target protein by IPTG concentration and induction time at different temperatures. MRANKL recombinant protein was purified in large quantities under the best conditions. The purified protein was filtered and diluted into different concentrations. The mRANKL protein stimulated the differentiation of mouse monocyte / macrophage Raw264.7 cells at the same time, and there was obvious osteoclast formation. The results showed that the recombinant protein of mRANKL had biological activity and could replace the commercial mouse RANKL.
【学位授予单位】：华东师范大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R363

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