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  本文選題：小鼠核因子κB受體活化因子配基(mRANKL) + 基因克隆　； 參考：《華東師范大學》2010年碩士論文

【摘要】： 核因子κB受體活化因子配基(RANKL)是誘導破骨細胞分化,成熟的重要因子,在生物學及醫(yī)學研究中具有廣泛的應用。RANKL主要結(jié)合到破骨前體細胞的核因子κB活化因子受體(RANK)上,啟動下游NF-κB,P38,ERK,JNK等信號通路,刺激破骨細胞的分化,成熟及成熟后的破骨細胞存活。由于RANKL在破骨細胞的發(fā)育過程中發(fā)揮重要作用,所以它的失調(diào)可引發(fā)多種骨骼疾病,如骨質(zhì)疏松,風濕性關節(jié)炎等疾病�，F(xiàn)在開發(fā)了多種針對RANKL及它所調(diào)控的信號通路的藥物,用于治療RANKL所引發(fā)的骨骼疾病。因此建立制取高純度的鼠源RANKL(mRANKL)重組蛋白,并誘導破骨細胞前體向破骨細胞分化這個模型具有重要的現(xiàn)實意義。它不僅可以為治療骨骼疾病的篩藥實驗提供模型,而且可以極大的降低實驗室成本。 在本實驗中,以鼠的骨髓細胞cDNA為模板,利用PCR技術體外擴增小鼠核因子κB受體活化因子配基(mRANKL)活性區(qū)cDNA,將PCR產(chǎn)物克隆至His標簽的融合蛋白表達載體pET28a(+),鑒定正確的質(zhì)粒轉(zhuǎn)化至BL21表達菌中。通過不同溫度、IPTG濃度及誘導時間誘導目的蛋白表達,篩選出mRANKL融合蛋白表達的最佳條件。以最佳條件大量純化mRANKL重組蛋白,純化后的蛋白過濾并稀釋成不同濃度,與商業(yè)購買的mRANKL蛋白同時分別刺激小鼠單核／巨噬細胞Raw264.7細胞分化,可以看見有明顯的破骨細胞形成,表明mRANKL重組蛋白具有生物活性,可替代商業(yè)產(chǎn)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.
【學位授予單位】：華東師范大學
【學位級別】：碩士
【學位授予年份】：2010
【分類號】：R363

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本文編號：2107247