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:目的 将肌腱蛋白 - R不同功能片段在原核中表达、纯化 ,并研究其初步的生物学功能 .方法 将编码肌腱蛋白 - R不同功能片段的表达质粒 p GEX- EGFL ,p GEX- EGFS,p GEX- FN1- 2 ,p GEX- FN6 - 8,p GEX- FG及空载 p GEX- KG转化入 E.Coli JM10 9中 .用 IPTG诱导表达 ,以谷胱甘肽琼脂糖为填充料进行亲合层析 ,纯化所表达的融合蛋白 .以纯化的融合蛋白作为培养液成分 ,GST为对照 ,观察纯化的肌腱蛋白 - R各功能片段对体外培养脊髓细胞活性及突起生长的影响 .结果 肌腱蛋白 R的 5个片段在大肠杆菌中都有表达 ,通过谷胱甘肽琼脂糖亲合层析 ,都得到了初步纯化 .通过对培养细胞进行 MTT染色 ,NSE染色后图像分析 ,结果表明 ,EGFL ,FN1- 2促进神经元的存活 ,EGFL ,EGFS和 FN6 - 8对突起的生长有明显的促进作用 .结论 肌腱蛋白 - R的不同功能片段可以通过大肠杆菌表达 ,经纯化获得蛋白 ,肌腱蛋白 -R不同的片段对体外培养的脊髓细胞有不同的作用
: Objective To express and purify different functional fragments of tendon protein-R in prokaryotes and to study their preliminary biological functions.Methods The expression plasmids pGEX-EGFL, p GEX-EGFS, p GEX-FN1-2, pGEX-FN6-8, pGEX-FG and empty pGEX-KG were transformed into E. coli JM109.The expression was induced with IPTG and the pro-glutathione sepharose The purified fusion protein was used as the culture medium and GST was used as a control to observe the effect of purified tenascin-R functional fragments on the activity of spinal cord cells and the growth of neurites in vitro.Results Tenascin The five fragments of R were expressed in E. coli and purified by glutathione agarose affinity chromatography.The MTT staining and NSE staining of the cultured cells were performed and the results showed that EGFL, FN1-2 promoted the survival of neurons, and EGFL, EGFS and FN6 - 8 significantly promoted the growth of neurites.Conclusion The different functional fragments of tenascin - R can be expressed in Escherichia coli, and the protein, tenascin R Different fragments have different effects on cultured spinal cord cells in vitro