大鼠透明质酸合成酶-3基因真核表达载体的构建及趋化作用研究

Construction of eukaryotic expression vector of rat HAS-3 gene and its effects on chemotaxis

目的:构建透明质酸合成酶-3(hyaluronan synthase-3)真核表达载体,转染真核细胞并检测重组蛋白酶活性。方法:提取损伤大鼠坐骨神经总RNA,RT-PCR扩增HAS-3编码框cDNA,构建于真核表达载体pcDNA3.1D中,并亚克隆到荧光载体pEGFP-N1中。将构建好的质粒转染大鼠施旺细胞RSC96,检测HAS-3的表达及酶活性。研究转染细胞培养上清对巨噬细胞的趋化作用。结果:HAS-3真核表达载体pcDNA3.1D-HAS3及pEGFP-HAS3测序结果显示片段插入正确,序列与GenBank公布数据一致,转染RSC96细胞并检测到重组蛋白的表达和酶活性,过表达HAS-3细胞的培养上清对巨噬细胞的趋化作用显著高于未转染细胞上清。结论:成功地构建HAS-3真核表达载体且真核表达的重组HAS-3具有合成HA活性,HAS-3过表达培养上清能趋化巨噬细胞,在体内可能参与到炎症反应中。

AIM： To construct the eukaryotic expression vector of rat HAS-3 gene,express it in RSC96 cells and detect the enzyme activity of the recombinant protein.METHODS： Rat HAS-3 gene was cloned from CCI rat injury nerve cDNA using RT-PCR and inserted into pcDNA3.1D and pEGFP-N1.The recombinant plasmids were transfected to RSC96 cells.The expression level and enzyme activities of the recombinant proteins were monitored.RESULTS： Rat HAS-3 gene was cloned into pcDNA3.1D and pEGFP-N1 correctly.Recombinant proteins were detected in RSC96 cells and the synthesis of HA was up-regulated after transcfection.The condition medium of RSC96 cells overexpressing HAS-3 had some effects on chemotaxis of macrophages.CONCLUSION： The eukaryotic expression vectors of rat HAS-3 has been constructed successfully.The transfection of HAS-3 gene to RSC96 cells is effective in chemotaxis of macrophages which demonstrates HAS-3 may contribute to the development of inflammation in vivo.