重组肌腱蛋白-R不同功能片段的表达、纯化和生物学功能

Expression and purification of recombinant tenascin R domains and their functions

: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

AIM To express and to purify recombinant tenascin R domains and to study their functions. METHODS The pGEX KG derived expression vectors which contained the DNA sequence encoding 5 domains of tenascin R weretransformed into E.coli JM109. The E.coli was then induced by isopropyl β thiogalactoside(IPTG). The expressed protein was purified with glutathione agarose beads. In order to study the effect of the purified proteins on survival and neurite growth of the spinal cord cells in vitro , the purified proteins were added in soluble form to the culture medium, using GST as a control. RESULTS Five tenascin R domains were expressed in E.coli and purified by glutathione agarose beads. The results showed that EGFL, FN1 2 promoted the survival of the cell and EGFL, EGFS, FN6 8 promoted the growth of neurite. CONCLUSION Recombinant tenascin R domains may express in E.coli and get the recombinant protein. The tenascin R domains have different effects on the spinal cord cell in vitro .