摘要： 目的   建立硫酸软骨素酶ABCⅠ（ChABCⅠ）的分泌型真核表达载体，并在胶质瘤细胞系中对其表达情况进行观察。方法   以真核表达载体pCDNA3.1/V5/HIS A 为载体，将基底膜40 蛋白信号肽编码区和ChABCⅠ成熟肽段编码区串联插入其多克隆位点，构建分泌型真核表达质粒pCDNA-BMS-CABCⅠ。采用该质粒转染人胶质瘤细胞系TJ905，培养3 d 后将培养液上清液行SDS-PAGE 和免疫印迹分析。结果   考马斯亮蓝染色显示有新条带出现，条带的相对分子质量大小与理论值一致，免疫印迹检测显示有V5 免疫反应性特异性条带出现。结论   该真核表达载体可介导硫酸软骨素酶ABCⅠ在神经胶质细胞来源的细胞中以分泌蛋白形式表达。

关键词: 软骨素硫酸酶类, 质粒, 转染, 神经胶质瘤, 免疫印迹法

Abstract: Objective To construct a secretary eukaryotic expression plasmid for chondroitinase ABCⅠ, and observe the expression of the target protein in a glioma cell line TJ905. Methods The DNA fragments encoding the signal peptide of basilar membrane 40 (BM40) as well as the mature fragment of chondroitinase ABCⅠ were inserted into the eukaryotic expression vector pCDNA3.1/V5/HIS A in series to construct the secretary eukaryotic expression plasmid pCDNA-BMS-CABCⅠ. Then the plasmid was used to transfect the glioma cell line TJ905, and after 3 d, the supernatant of the culture media was collected and analysed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. Results On polyacrylamide gel electrophoresis (PAGE) gel, a new band corresponding to the theoretic molecular weight was spotted. Western blotting showed a specific band of V5 immunoreactivity. Conclusion The newly constructed plasmid can mediate the eukaryotic expression of chondroitinase ABC Ⅰ in secretary manner.

Key words: Chondroitinsulfatases, Plasmids, Transfection, Glioma, Immunoblotting