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

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. DOI：10.3969/j.issn.1672-6731.2010.04.018

Keywords

• Chondroitinsulfatases
• Plasmids
• Transfection
• Glioma
• Immunoblotting