硫酸软骨素酶ABCⅠ分泌型真核表达质粒的构建

The construction of a secretary eukaryotic expression plasmid of chondroitinase ABC Ⅰ

目的建立硫酸软骨素酶ABC Ⅰ(ChABC Ⅰ)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ChABC Ⅰ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ABC Ⅰ在神经胶质细胞来源的细胞中以分泌蛋白形式表达。

Objective To construct a secretary eukaryotic expression plasmid for chondroitinase ABC Ⅰ , 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 ABC Ⅰ were inserted into the eukaryotic expression vector pCDNA3.1/V5/HIS A in series to construct the secretary eukaryotic expression plasmid pCDNA-BMS-CABC Ⅰ . 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- polyacrytamide get 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.